The advent of combination antiretroviral therapy (cART) has largely eliminated the incidence of encephalitis-like in- ?ammation in human immunode?ciency virus-1 (HIV-1) patients; however, HIV-1-associated neurocognitive disorder (HAND) persists in virologically suppressed patients. HAND is observed in 50% of HIV-1-infected individuals in coun- tries with widespread access to cART . Recent data obtained from humanized mouse models of HIV-1 infection indi- cate that tissue macrophages are a long-lived HIV-1 reservoir even under ef?cacious cART [2, 3], suggesting that tissue macrophages, including microglia and splenic macrophages, can drive chronic in?ammation in their resident tissues. Concurrently, next-generation sequencing technologies have vastly expanded our knowledge of factors that regulate the development and homeostasis of tissue macrophages (e.g., microglia, splenic macrophages, etc) [9, 10, 11], permitting an examination of their relevance to disease processes. Among these is the master regulator transcription factor Mafb, which has been shown to contribute to the expression of tissue macrophage-associated transcripts  as well as the repression of anti-viral and immune-related genes . My preliminary data shows that Mafb overexpression in the BV2 microglial cell line upregulates the tissue macrophage-associated transcripts Mertk and C1qa, while significantly reducing TNF-a and CCL4 production in response to stimulation with HIV-1 Tat. The primary goal of this proposal is to determine mechanistically how Mafb suppresses pro-in?ammatory signaling in microglia and whether the MLK3 inhibitor URMC-099 promotes the stability of the Mafb protein under in?ammatory conditions. To this end, I propose two aims. First, I will use an inducible CRISPR/Cas9-based activation (CRISPRa) approach to titrate Mafb expression in BV2 mouse microglia and in C20 human microglia cell lines to determine whether Mafb gene dosage is inversely correlated TNF-a and CCL4 expression at both the mRNA and protein levels. I will determine whether Mafb acts as a transcriptional suppressor at these loci using chromatin immunoprecipitation in Mafb-overexpressing microglia and lu- ciferase reporter assays in HEK293T cells. Second, I will determine whether inhibition of c-Jun N-terminal kinase (JNK) prolongs the half-life of Mafb protein following HIV-1 Tat stimulation of microglia in vitro and in tissue macrophages (including microglia and splenic macrophages) in vivo using HIV-1 infected humanized NOD/SCID/IL2Rgc-/- (hNSG) mice. Altogether, the proposed experiments will identify key roles for Mafb in de?ning features of tissue macrophage physiology, particularly within the context of chronic HIV-1 infection. Accomplishment of these goals may also inform therapeutic strategies for a wide variety of in?ammatory conditions in which tissue macrophages contribute to collateral tissue damage and worsened disease outcomes.
HIV-1 associated neurocognitive disorder (HAND) persists despite combination antiretroviral therapies. The proposed research will examine a novel mechanism through which the MLK3 inhibitor URMC-099 suppresses HIV-1 Tat-induced microglial activation by promoting the stability of the tissue-macrophage-associated transcription factor Mafb. Given the enrichment of Mafb across tissue macrophage subsets, the ?ndings generated by the proposed experiments may have broad implications for addressing in?ammatory tissue damage outside of the CNS of chronically infected HIV-1 patients.
|Miller-Rhodes, Patrick (2018) CX3CR1 Does Not Universally Mediate Microglia-Neuron Crosstalk during Synaptic Plasticity. J Neurosci 38:4457-4459|