Cytoplasmic HIV-RNA in monocytes determines microglial activation and neuronal cell death in HIV-associated neurodegeneration

Abstract

Despite highly active antiretroviral therapy, HIV-associated neurocognitive disorders (HAND) are still highly prevalent. Direct neurotoxicity of microglia activated by HIV-infected monocytes independent from viral replication may account for this observation. To investigate underlying molecular and viral determinants, human monocytoid cells (U937) transduced with HIV-particles were co-cultured with primary human microglia or astrocytes. Using genetically-engineered HIV-particles key steps of infection were examined. Levels of pro-inflammatory/neurotoxic cytokines were investigated in co-culture supernatants by flow cytometry. Neurotoxicity mediated by the supernatants was analysed using primary cortical rat neurons. To corroborate our findings, cytokine profiles in cerebrospinal fluid (CSF) of neuropsychologically asymptomatic HIV positive (HIV(+)) patients (n=45) were correlated with neurofilament H (NfH) as surrogate of neuronal/axonal degeneration. In contrast to direct exposure of HIV to microglia, only the presence of HIV-transduced monocytoid cells strongly activated human microglia as evidenced by enhanced secretion of CXCL10, CCL5, CCL2, and IL-6 (1.3-7.1-fold; p<0.01) leading to two-fold increased neurotoxicity (p<0.001). In direct comparison, astrocyte activation by HIV-transduced monocytoid cells was limited. Using different mutant HIV-particles we show that the presence of cytoplasmic HIV-RNA in monocytoid cells is the viral determinant for this unique microglial activation pattern and subsequent neuronal cell death; reverse transcription and expression of viral genes were not essential. In CSF of presymptomatic HIV(+) patients, CXCL10, CCL5 and IL-6 were correlated with NfH as surrogate marker of neurodegeneration as well as CSF-pleocytosis. In conclusion, cytosolic viral RNA in monocytes is mandatory for subsequent microglial activation and neurotoxicity; activated astrocytes may augment neuroinflammation. In addition, neuroinflammation and neurodegeneration occur even in preclinical HIV(+) patients and are associated with cytokines regulated in vitro. Our data may aid in the development of biomarkers and glia-directed therapeutic approaches of HAND.

Keywords: Cytokines; Glial activation; HIV RNA; HIV related neurological diseases; Microglia; Neurodegeneration; Neuroinflammation.