Neurokinin-1 Receptor (NK1-R) Expression in the Brains of SIV-Infected Rhesus Macaques : Implications for Substance P in NK1-R Immune Cell Trafficking into the CNS

Recent studies suggest a link between neuropsychiatric disorders and HIV/SIV infection. Most evidence indicates that monocytes/macrophages are the primary cell type infected within the CNS and that they contribute to CNS inflammation and neurological disease. Substance P (SP), a pleotropic neuropeptide implicated in inflammation, depression, and immune modulation via interaction with its cognate receptor, the neurokinin 1 receptor (NK1-R), is produced by monocyte/macrophages. While the presence of NK1-R on neurons is well known, its role on cells of the immune system such as monocyte/macrophages is just beginning to emerge. Therefore, we have examined the expression of SP and NK1-R and their relationship to SIV/HIV encephalitis (SIVE/HIVE) lesions and SIV-infected cells. These studies demonstrated intense expression of SP and NK1-R in SIVE lesions, with macrophages being the principal cell expressing NK1-R. Interestingly, all of the SIV-infected macrophages expressed NK1-R. Additionally, we examined the functional role of SP as a proinflammatory mediator of monocyte activation and chemotaxis. These studies demonstrated that treatment of monocytes with SP elicited changes in cell-surface expression for CCR5 and NK1-R in a dose-dependent manner. Moreover, pretreatment with SP enhanced both SP- and CCL5-mediated chemotaxis. All of these findings suggest that SP and NK1-R are important in SIV infection of macrophages and the development of SIVE lesions.The neuropathogenesis of HIV infection is complex and has led to the development of nonhuman primate models using infection of macaques with the simian immunodeficiency virus (SIV). The human and simian immunodeficiency viruses are closely related and produce nearly identical conditions in their respective hosts, thus the rhesus macaque infected with SIV is the premier animal model for the study of AIDS pathogenesis in general and of the neuropathogenesis of AIDS in particular. Additionally, nonhuman primates are widely used in neuroscience research, including neurophysiologic and neurobehavioral studies. Recent studies suggest a link between psychiatric disorders like stress and depression and the neuropathogenesis of HIV and/or its progression to AIDS.^1,2 Neuroinvasion by SIV and HIV occurs early in infection at the time of peak viremia. The virus most likely enters the brain within cells of monocyte/macrophage lineage, and the perivascular macrophage is the primary cell type productively infected within the CNS.^3,4Approximately 25 to 30% of untreated HIV-infected adult humans develop a debilitating neurological disorder termed AIDS dementia complex (ADC).^5,6,7 A wide variety of cytokines, which include IL-1beta, IL-6, and TNF-α, chemokines (CCL2, CCL3, CCL4 and CCL5), adhesion molecules, and other molecules such as Substance P (SP) produced by different cell types have been implicated in this process.^1,4,8,9 Microscopic evaluation of brains from individuals with ADC reveals a broad spectrum of pathological features including neuronal changes, multifocal encephalitis, accumulation of inflammatory macrophages and multinucleated giant cells (MNGCs), cerebral cortical atrophy, and white matter pallor.^5,10 The histopathological substrate of ADC, referred to as HIV encephalitis (HIVE), is characterized by perivascular accumulation of macrophages and MNGCs in the CNS with abundant infection of brain macrophages.^4,11 In addition to the pathological manifestations within the brains of individuals with ADC, extensive neurobehavioral effects have been characterized and include impaired fine motor control and memory, altered emotional control, motor slowing, and possibly depression. Compelling data from recent studies reveal the significance of inflammatory mediators including the neuropeptide SP in the neuropathogenesis of AIDS^12,13,14 as well as in clinical neurobehavioral effects of depression.^2,3,4 Such findings suggest that SP participates in important aspects of immune-neural communication and likely contributes to immune modulation in HIV/SIV infection.SP is an 11-aa neuropeptide and is the most extensively studied and most abundant member of the tachykinin family. SP is synthesized mainly by primary sensory neurons, however recent studies have demonstrated that immune cells also express SP mRNA and protein during SIV/HIV-infection,^1,8 suggesting that it may contribute to neurological disease by acting on its preferred receptor, NK1-R, a G-protein–coupled receptor expressed on T-cells, B-cells, monocyte/macrophages, NK cells, astrocytes, and neurons.⁹ Activation of NK1-R by SP results in increased phagocytic response in macrophages, enhanced inflammatory cytokine production by immune cells, and possible induction of a chemotactic response in monocyte macrophages, thus facilitating immune cell trafficking at sites of inflammation or infection.^{16,17,18,19,20} SP has a role in AIDS, and results from recent in vivo studies^1,8,15,21,22 revealed that NK1-R antagonists have an antiviral effect,²³ likely through down-regulation of CCR5⁹ as well as immunomodulatory and antidepressive effects.^23,24 The major focus of existing work on SP (and its receptor-NK1-R) in macaques, however, has focused on its role as a neurotransmitter or neuromodulator and therefore the distribution of SP and NK1-R on neurons is well known, while little data exists on the in vivo distribution of SP and NK1-R on other cell types.Because the cellular expression of SP and its receptor NK1-R is not fully characterized in normal or SIV-infected macaques, we examined the cellular distribution/location and phenotype of SP and NK1-R expressing cells in the CNS in vitro and in vivo using multiple techniques and at various stages of infection in animals with or without SIVE. Such analysis allowed us to assess spatial correlations between the presence of SIV-infected cells and cells positive for SP and NK1-R. Additionally, we examined the functional aspects of SP/NK1-R signaling in monocyte activation and chemotaxis. Data from our immunofluorescence and in situ hybridization studies show that while NK1-R is expressed in astrocytes and neurons, it is intensely expressed in SIVE lesions. NK1-R expression was detected in all SIV-infected cells associated with SIVE lesions, of which macrophages were the predominant cell type and also the primary cell type expressing NK1-R. T-cells were found in small numbers in SIVE lesions and were rarely infected but were shown to express NK1-R when present. Our quantitative flow cytometric analysis demonstrates that SP functionally regulates NK1-R and CCR5 expression on macaque monocytes. Furthermore, pretreatment of monocytes with SP enhanced SP-mediated chemotaxis as well as CCL5-mediated chemotaxis. The enhancement of SP-mediated chemotaxis in the presence of CCL5 demonstrates cross talk between NK1-R and CCR5 signaling pathways. These findings suggest that SP contributes to SIV-associated neurological disease and suggest that SP also has a role in augmented cellular trafficking across the blood-brain barrier and thus the development of SIVE lesions. This insight into the function of SP and NK1-R allows for a better understanding of the interaction between the immune and nervous systems, and may lead to advancements in treatment of neurological and neuropsychiatric disease.