Increased Expression of Monocyte CD44v6 Correlates with the Development of Encephalitis in Rhesus Macaques Infected with Simian Immunodeficiency Virus

Maria Cecilia G. Marcondes 0 Caroline M. S. Lanigan 0 Tricia H. Burdo 0 Debbie D. Watry 0 Howard S. Fox () 0 0 Molecular and Integrative Neurosciences Department, The Scripps Research Institute , La Jolla, California In people infected with human immunodeficiency virus type 1 (HIV-1), the accumulation of macrophages in the brain correlates with encephalitis and dementia. We hypothesized that a pattern of surface marker expression in blood monocytes may serve as a marker for central nervous system (CNS) disease. Using the simian immunodeficiency virus (SIV)-rhesus monkey model, we analyzed functionally relevant surface markers on monocytes and macrophages from the blood and brain in animals that did or did not develop SIV encephalitis. At necropsy, multiple markers (CD44v6, CCR2, and CCR5 on blood monocytes and brain microglia and/or macrophages, and CX3CR1 on blood monocytes) allowed us to distinguish animals with encephalitis from those without. Furthermore, the level of expression of CD44v6 on the 2 main populations of blood monocytes-those that express either low or high levels of CD16 -was significantly increased in animals with encephalitis. A longitudinal analysis of blood monocyte markers revealed that as early as 28 days after inoculation, CD44v6 staining could distinguish the 2 groups. This provides a potential peripheral biomarker to identify individuals who may develop the HIVinduced CNS disease. Furthermore, given its role in cellular adhesion and as an osteopontin receptor, CD44v6 upregulation on monocytes offers functional clues to the pathogenesis of such complications, and provides a target for preventative and therapeutic measures. - Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) are retroviruses that disturb immune system integrity in their respective hosts and induce variable levels of central nervous system (CNS) dysfunction. The most severe CNS disease is characterized pathologically by the presence of encephalitis, dominated by macrophages in the brain, and it is clinically characterized by debilitating dementia. In the brain, virus is found predominantly in macrophages, as well as in microglia. Although the etiopathogenesis of HIV-induced CNS disorders are still under active investigation, resident microglia and infiltrating brain macrophages can secrete harmful products [13] that are detrimental to CNS function. In addition, viral proteins themselves have been demonstrated to have numerous neuroinjurious properties. Although efficacious therapy is now available in developed countries, resulting in a reduced incidence of CNS disease and other complications of HIV infection, the prevalence of CNS-related abnormalities has not decreased, and in fact appears to be increasing [4, 5]. In addition, the CNS can still serve as a reservoir for the virus. The discovery of biomarkers to aid in the identification of individuals at risk for, or in the process of developing, CNS disease in the setting of HIV infection has been a challenge. So far, markers have not been found to correlate with encephalitic outcome. The accumulation of macrophages in the brain has been the best correlate of CNS disease; however, this is identified post mortem. Therefore, the detection of blood monocyte markers modulated by the disease state that correlate with pathological brain findings can be relevant for diagnosis. Monocytes are a heterogeneous population of cells, in terms of the expression of surface markers [6]. One major subdivision of this cell population, monocytes that are CD16 (as opposed to CD16low), account for to 5%10% of monocytes, but this population is expanded in several pathological conditions [6]. For instance, patients with symptoms of HIV-induced CNS disease and monkeys with symptoms of SIV-induced CNS disease were found to have increased CD16 monocytes in the blood [79]. Furthermore, CD16 monocytes have been suggested to be the cell type that infiltrates the CNS in correlation with HIV dementia, because the perivascular macrophages in the brain are CD16 [10 12]. Altered monocyte migration into the brain leads to the encephalitis caused by lentiviruses, including HIV and SIV. In the SIV-monkey model, subpopulations of monocytes are expanded during acute SIV infection, but this expansion was not unique or greater in macaques that developed encephalitis [13]. Furthermore, the proportion of monocytes that expressed markers such as CD62L, HLA-DR, CD64, and CD40 varied among animals that eventually developed encephalitis [14]. Although no correlation was found, this does not rule out either the importance of activated monocytes in the development of CNS inflammation or the possibility that a correlative marker for CNS disease could be found on monocytes. Therefore, the search for predictors remains a challenge. Similar to humans with dementia and HIV-induced encephalitis, SIV-infected monkeys with neurological symptoms frequently prove to have encephalitis. Therefore, the use of the monkey-SIV model can aid in the detection of a pattern of markers that can be evaluated prior to the development of disease. Here, we compared surface markers expressed on blood monocytes and tissue macrophages and identified patterns that characterize animals with encephalitis. We report the identification of a candidate biomarker, CD44v6, whose expression on peripheral monocytes is correlated with the development of encephalitis. MATERIALS AND METHODS Monkeys. Rhesus macaques were infected with a microgliapassaged stock of SIVmac251 [15], and sacrificed 6 months after infection (or earlier, if they exhibited symptoms of disease, which were determined using criteria that included progressive weight loss, marked hematological abnormalities, persistent anorexia, diarrhea not responsive to treatment, and the presence of neurological or behavioral signs). Necropsies were performed after the administration of ketamine (50 mg/kg), xylazine (4 mg/ kg), and pentobarbital (10 mg/kg), and animals were extensively perfused with sterile PBS containing 1 U/mL heparin to flush bloodborne cells from the organs. All organs were examined grossly and microscopically. Encephalitis was diagnosed by the presence of multinucleated giant cells, perivascular and infiltrating macrophages, and the expression of viral RNA (as determined by in situ hybridization) and/or protein (as determined by immunohistochemical analysis) in the brain. Two groups of animals were thus identified histopathologically: those that developed encephalitis (SIVE animals, n 6) and those that did Table 1. Expression of surface functional markers in monocytes and/or macrophages in the blood and brain of macaques infected with simian immunodeficiency virus. Marker, SIV encephalitis status Date are the mean percentage of monocytes and/or macrophages expressing markers SD. Cells were stained for flow cytometry and gated on CD14 , CD16low, and CD16 cells. The results are expressed in percentage with (...truncated)