Lesions are composed of lymphocytes and macrophages. Double immunolabeling and spectral imaging with pseudofluorescence coloring identifies CD4 + T-lymphocytes and macrophages (red) within inflammatory foci (B). Expression of CD3 (green) identifies lymphocytes within inflammatory foci (A). Nuclei are stained with Mayer’s hematoxylin (blue) (C). CD4 + lymphocytes express both CD4 and CD3 molecules (arrows) (D). Note CD3 expression on cells that do not express CD4 (arrowheads). doi:10.1371/journal.pone.0014429.g005 

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... using a bead beater and 1 mm diameter silica beads (Biospec Products Inc., Bartlesville, OK). After homogenization, the homogenate was removed to a new tube, 0.2 volumes of chloroform was added, and the aqueous phase was collected after centrifugation at 8,000 6 g for 5 minutes at 4 u C. The aqueous phase was combined with an equal volume of 70% ethanol, one ml of 4M guanidinium isothiocyanate was added, and the mixture was loaded onto a RNeasy spin column (Qiagen). After on-column DNase treatment (Qiagen), the total RNA was eluted in RNase free water, 2 units/ m l of recombinant RNase inhibitor (RNasin, Promega) was added, and the RNA was frozen at –80 C. The amount of RNA extracted from tissue specimens was measured using the Quant-iT Ribo Green RNA assay (Invitrogen), adhering to the manufacturer’s instructions, and measuring fluorescence for 0.1 seconds at excitations and emissions of 485 nm and 535 nm, respectively, using a Victor 3 V 1420 Multilabel Counter (Perkin Elmer, Waltham, MA). The absolute quantification of SIV Gag, TNF- a and RPL13A transcripts in total RNA from left ventricle and spleen specimens was conducted using TaqMan probes and TaqMan One-Step RT-PCR master mix reagents (Applied Biosystems, Foster City, CA) on an ABI Prism 7700 thermal cycler (Applied Biosystems). Total RNA from myocardial tissues (100 ng per specimen) was combined with 200 nM each of forward and reverse primers, 1x One-Step RT-PCR master mix and, 100 nM of TaqMan probe in a 50 m l reaction. For use as internal RNA standards, known copy numbers of RNA transcripts were serially diluted in RNase free water from 10 6 through 10 1 copies. Amplification data were analyzed using Sequence Detection Version 1 software (Applied Biosystems). To investigate the expression of cytokines and chemokines in myocardial tissue, 20 ng of cDNA from each specimen was used as template in a 25 m l reaction containing 1x SYBR Green master mix (Applied Biosystems) and forward and reverse primers (100 mM each). Reactions were conducted in duplicate on an ABI 7500 thermal cycler, with the following conditions: 1 cycle of 96 C for 2 minutes; 45 cycles of 96 C for 30 seconds and 60 C for 1 minute followed by one cycle of dissociation from 96 u C to 60 u C. At the end of each run, the data were analyzed using Sequence Detection Version 1 software (Applied Biosystems). Fold-regulation of expression of each gene was calculated from the Ct values after normalizing with the Ct values obtained for RPL13A mRNA, the internal control gene. The primers used in the study are shown in Table 3 . A two-tailed, nonparametric Mann-Whitney U test was used to compare the copy numbers of SIV and TNF- a mRNA transcripts, which are reported as median values 6 semi-interquartile range. Significant differences were assumed for probability values of p , 0.05. Histopathological examination of routine H&E sections revealed multifocal infiltrates of mononuclear inflammatory cells (morphologically consistent with macrophages and lymphocytes), which were dissecting and compressing cardiomyocytes in sections of myocardium from all four animals in the CART group. Necrosis and degeneration of cardiomyocytes was apparent within the lesions, with no evidence of fibrosis ( Figure 1 ). In addition, reactive, hypertrophic endothelial cells were observed bulging into vascular lumens throughout the myocardial sections of 3 of the 4 animals that received CART. In contrast, no degenerative changes or significant inflammatory foci were present in the myocardial sections from any animals in either the control group or the untreated SIV positive animals with simian AIDS. The absence of intralesional fibrosis in the myocardium of affected animals was confirmed by Masson’s trichrome stain, which revealed that there was no significant difference in the amount of collagen present within the myocardium of animals among all three groups ( Figures 2A and 2B ). The pericardium was normal in all animals. IHC was used to characterize the inflammatory infiltrates in the myocardium. Inflammatory foci were composed largely of Iba-1 expressing cells, compatible with macrophage lineage; moreover, 60–80% of the Iba-1 positive cells also expressed the CD68 glycoprotein, suggestive of an activated phenotype (Figure 3) . IHC failed to reveal significant expression of CD8 (which is + expressed by both CD8 T lymphocytes and natural killer (NK) cells) or CD20 (which identifies B lymphocytes) within inflamma- tory foci ( Figures 4A and 4B ). Because small numbers of CD3 cells had been localized within the inflammatory foci by single- label IHC in the absence of significant CD8 expression, multiparameter IHC and spectral imaging were used to defini- tively identify the phenotype of intralesional T lymphocytes. Dual- label IHC for CD3 and CD4 expression revealed small numbers of + + CD3 /CD4 double-positive lymphocytes and less frequent CD3 + /CD4 - cells, which were assumed to be CD3 + /CD8 + T lymphocytes ( Figure 5 ). Taken together, these results indicate that the inflammatory foci were primarily composed of activated + macrophages and small numbers of CD4 positive helper T lymphocytes. Despite the abundance of activated macrophages in the myocardial lesions, expression of pro-IL-18, the inactive precursor of the proinflammatory cytokine IL-18, was confined to faint cytoplasmic reactivity in a minority of the macrophages present in the inflammatory foci ( Figures 4C and 4D ). ISH for SIVmac239 RNA revealed very few productively infected cells in the myocardium of either untreated animals or those that received CART ( Figures 2C and 2D ). However, quantification of viral RNA by TaqMan real time RT-PCR revealed a significantly lower virus burden in the myocardium of animals that received CART as compared to the myocardium of untreated animals (median values of 4.10 6 0.18 versus 4.85 6 0.09 log 10 SIV copies/ mg, respectively; p , 0.05) (Figure 6A) . These results indicate that the myocardium is not a primary target organ for virus replication in CD8 lymphocyte-depleted animals, and suggest that the lesions present in the myocardium of animals in the CART group are not directly associated with SIV infection. In addition to lower myocardial tissue virus burden, CART was associated with a significantly lower terminal plasma virus load ( Table 1 ). We quantified mRNA transcripts for the proinflammatory cytokine TNF- a in myocardial tissue from all animals in the control, untreated and CART groups. Significantly greater levels of TNF- a mRNA expression were measured in the myocardium of animals that received CART than in myocardial tissue from the untreated or control macaques (4.05 6 0.19 vs. 3.45 6 0.10 vs. 3.25 6 0.20 log TNF- a copies/mg, respectively; p , 0.05) ( Figure 6B ). There was no difference in the quantity of TNF- a mRNA transcripts measured in myocardial tissue from untreated versus control groups. Since macrophages were the principal cellular component of the inflammatory and degenerative myocardial lesions observed in animals that received CART, and significantly higher levels of TNF- a mRNA expression had been measured in myocardial specimens from the CART group, we sought to measure the levels of mRNA expression for other key proinflammatory cytokines and chemokines that have been implicated in the pathogenesis and regulation of cytotoxic injury. To that end, we used SYBR Green real time RT-PCR and gene specific primers to measure the quantity of several mRNA transcripts, including IL-1 b , IL-6, IL- 10, IL-18, INF- c , MCP-1, CXCL9 and CXCL11 in frozen specimens of myocardial tissue from all three groups of animals. Myocardial tissue from control animals expressed very low levels of IL-1 b , Il-6, IL-10, IL-18, CCR5 and INF- c mRNA, with cycle threshold (Ct) values ranging from 33-37 (data not shown). In contrast, appreciable levels of MCP-1, CXCL9, CXCL11 and ICAM-1 mRNA were detected in controls, with Ct values ranging from 27-31, indicating an abundance of expression of these genes in the myocardium. Table 4 shows the expression patterns of cytokines in the myocardium from untreated and CART groups of SIV-positive monkeys as compared to the SIV-negative CD8- depleted control group. Higher levels of CCR5, CXCL11, INF- c , and IL-1 b mRNA were expressed in the myocardium of SIV- positive animals, regardless of whether they received antiretroviral therapy, although the quantity of CXCL11 mRNA expressed in SIV-infected, untreated macaques was more than two-fold greater than in SIV-positive animals that received CART. The level of IL- 18 mRNA expression was lower in both treated and untreated SIV-positive macaques, while IL-6 mRNA levels were similar to SIV-negative controls. Interestingly, while the quantities of IL-10, ICAM-1 and MCP-1 mRNA were similar in myocardial specimens from SIV-positive macaques that received CART and SIV-negative controls, greater quantities of all three transcripts were measured in myocardial specimens from SIV-infected, untreated animals than in the other two groups. In contrast, while myocardial CXCL9 mRNA levels were similar between SIV-negative animals and SIV-positive monkeys that received CART, the quantity of myocardial CXCL9 transcripts was 7-fold lower in untreated SIV-infected animals. With regard to the relative magnitude of CXCL9 and ICAM-1 mRNA expression, differences between treated and untreated SIV-positive macaques correlated precisely with the pattern of protein expression in tissue sections by immunohistochemistry. Strong expression of CXCL9 (which is induced by IFN- c ) was observed by the macrophages present in the inflammatory foci ( Figures 4B, 4C and 4D ) in the myocardium of animals that received CART, while CXCL9 expression was not detected in sections of myocardium from untreated monkeys. Surprisingly, the expression of ICAM-1, which is induced by TNF- a , was significantly weaker in myocardial sections from animals that received CART as ...