The temporal and stimuli-specific effects of LPS and IFNγ on microglial activation

Microglia, the resident immune cells of the central nervous system (CNS), play a pivotal role in health and disease maintaining homeostasis and mediating neuroinflammatory responses. Their activation is a dynamic and context-dependent process characterized by diverse phenotypic states defined by transcriptomic, proteomic, and morphological characteristics. While lipopolysaccharide (LPS) is widely used as an inflammatory stimulus in microglial research, its physiological relevance remains debated. Interferon gamma (IFNγ), a key pro-inflammatory cytokine involved in immune priming, more closely mimics CNS inflammatory conditions. In this study, we systematically investigated the temporal activation profiles of human iPSC-derived microglia (hiMG) in response to LPS, IFNγ, and their combination. Transcriptomic analysis at 24 h revealed robust differential gene expression, with over 7,000 genes altered by LPS and more than 8,500 by LPS/IFNγ co-stimulation. These profiles partially overlapped with disease-associated microglia (DAM) signatures, including upregulation of <i>S100A9</i>, <i>CD44</i>, <i>ACSL1</i>, and <i>HIF1A</i>, and downregulation of <i>TREM2</i>, <i>GPNMB</i>, <i>FABP3</i>, <i>LGMN</i>, and <i>LPL</i>. Cytokine expression changes were detectable as early as 1 h post-treatment, predominantly following LPS exposure, and displayed distinct early (≤2 h), mid (4-12 h), and late (24-96 h) temporal patterns. IFNγ alone induced modest transcriptomic and cytokine responses but contributed to sustained inflammatory signatures when combined with LPS. Morphological analysis showed marked LPS- and LPS/IFNγ-induced structural remodeling of hiMG consistent with activation. To assess protein-level dynamics, targeted mass spectrometry quantified secreted ApoE, CD44, FUCA1, Galectin-3, and Osteopontin, all relevant to microglial activation, which were compared to cellular protein expression measured by western blot. Time-dependent increases were most prominent following LPS and LPS/IFNγ treatment, although secreted Osteopontin levels were highest with IFNγ alone, highlighting stimulus-specific effects. Collectively, these data demonstrate that microglial activation is highly time- and stimulus-dependent, with LPS eliciting the strongest responses, and IFNγ modulating these effects. Our findings underscore the importance of temporal resolution in modeling microglial activation and provide insight into the mechanistic underpinnings of microglial activation relevant to neurodegeneration and therapeutic targeting.