Abstract
<jats:p>Background A significant number of people with HIV-1 still experience neurocognitive impairments (NCI), despite effective antiretroviral treatment. HIV-NCI is diverse and multifactorial, with mechanisms that cause its development and progression still not fully understood. We examined early HIV-related changes in brain stability and studied neuroimmune interactions at the single-cell level to better understand how NCI develops. Methods To model changes in brain homeostasis, we developed an advanced human iPSC-derived 3D cerebral assembloid model that includes microglia, by co-developing neural progenitor cells with tdTomato-tagged and CD34+ cell-derived microglial precursors. Assembloids were infected with a macrophage R5-tropic HIV-1 strain NL-AD8. Viral spread was measured using a proviral DNA assay, qPCR for HIV RNA, and 3D immunostaining for Tat protein. Single-cell transcriptomics with tdTomato lineage tracing revealed HIV-1 induced disturbances and cell-type-specific responses. The niche net algorithm was used to identify ligand-receptor interactions between microglia and the brain microenvironment during homeostasis and HIV infection. Results Highly ramified tdTomato+ IBA-1+ microglia were evenly distributed throughout the assembloids within 15 days of culture. Single-cell transcriptomics identified microglia, excitatory/inhibitory neurons, astrocytes, and oligodendrocyte precursors within the assembloids. Neurons in microglia-containing assembloids upregulated genes related to neurotransmission, synaptogenesis, and neuronal development compared to neurons in organoids without microglia. Niche net analysis showed microglia-derived neurotropic ligands supported neuronal and astrocytic differentiation. The R5-tropic HIV-1 specifically targeted microglia, inducing a reactive phenotype that transmitted interferon and pro-inflammatory signals to nearby cells and increased MHC-I antigen-presentation genes. Notably, neuroprotective ligands from non-glial cells and bystander microglia in the assembloids attempted to counteract HIV-related inflammation and promote neural repair. Conclusions Our microglia-containing assembloid model replicates in vivo neurodevelopmental interactions, allowing high-resolution analysis of homeostatic and HIV-induced responses across different brain cell types. Homeostatic microglia support neuronal health, while HIV infection triggers a reactive state that spreads inflammatory signals within the brain environment. The presence of multiple glial and non-glial populations uncovered previously unknown crosstalk, including bystander microglial phenotypes and neuroprotective signaling mechanisms that counteract inflammation. These findings emphasize early HIV responses that balance injury and adaptation, offering insights for developing therapies that target microglial activation, boost neuroprotection, and address HIV reservoirs in the brain.</jats:p>