Introduction
Microglia dynamically regulate their phenotype in response to brain microenvironment conditions, with this changing their mediation of inflammatory and reparative processes. Hypoxic signalling is a central driver of microglial reprogramming, particularly in the context of neurodegenerative disease. Key mediators of this response include hypoxia-inducible factor 1 alpha (HIF1α) and Toll-like receptor 4 (TLR4), which coordinate immune adaptation under low oxygen conditions. However, the molecular crosstalk between HIF1α, TLR4, and subsequent functional responses remains poorly defined.

Aim

This study aims to understand microglial behaviour in hypoxic niches and its implications for inflammation within the neural environment and tissue repair.

Methods
All animals for this research were sacrificed by pentobarbitol injection via (Animals (Scientific Procedures) Act, 1986) (ASPA) compliant Schedule 1 procedures. Work was performed with Keele University ethics committee approval under establishment licence X350251A8.

This study examined the inflammatory and functional responses of primary microglia from postnatal mouse brains (P1-P3) and BV-2 cells under normoxic (21% O₂) and hypoxic (1% O₂) conditions, following 24h stimulation with a range of LPS concentrations (0–10,000 ng/mL). To investigate HIF1α involvement, cultures were simultaneously treated with the prolyl hydroxylase inhibitor FG4592 (Roxadustat). Effects on TNF-α production, metabolic activity, and cytotoxicity were respectively assessed by ELISA, MTT, and LDH assays. Phagocytic function was analysed using fluorescent nanoparticle uptake, and gene expression of pro-inflammatory and anti-inflammatory markers including TNF-α, IL-6, Arg1, and TLR4 assessed by qPCR.

Results
Primary microglia displayed by mean ~49% higher TNF-α  concentrations than BV-2 cells across all concentrations ≥100 ng/mL. Hypoxia suppressed TNF-α production in both cell types, and reduced microglial viability and phagocytosis in BV-2 cultures. FG4592 sensitised primary microglia to low-dose LPS (1–10 ng/mL); increasing TNF-α output at these doses without doing so at greater LPS concentrations. This sensitisation was not observed in BV-2 cells, where FG4592 reduced TNF-α release in a manner comparable to hypoxia. HIF1α stabilisation increased IL-6 and Arg1 expression in BV-2 cultures. Additionally, it did not significantly augment the phagocytotic activity of primary cells. Taken in their totality, these results suggest that while HIF1α contributes to microglial pro-inflammatory cytokine production, it does so though context-dependent regulation of microglial function.

Conclusions
This study demonstrates that hypoxia and HIF1α stabilisation do not universally enhance microglial pro-inflammatory responses. Instead, pro-inflammatory microglial function is suppressed by hypoxia, while HIF1’s effects are cell-line specific and sensitive to the intensity of inflammatory stimuli. BV-2 cells showed diminished capacity for inflammatory activation and adaptation under hypoxic stress, while primary microglia demonstrated increased sensitivity to low-level inflammatory cues when HIF1α was stabilised. These findings highlight the limitations of immortalised microglia as proxies for primary cell models. They suggest that therapeutic modalities targeting HIF1α are unlikely to exacerbate already highly inflammatory microenvironments, such as in in stroke, but may bias microglia towards greater TNF- α release overall.