IRF5-RELATED PROINFLAMMATORY ACTIVATION OF MICROGLIAL CELLS IS COUNTERACTED BY NRF2/HO-1

BACKGROUND-AIM Microglia are innate immune cells residing in the central nervous system, their dysregulated activation leads to neuroinflammation and neuropathologies through molecular mechanisms not well understood yet. Several studies have highlighted the transcription factor IRF5 as a major player in inducing inflammation through an interaction with the NF-κB pathway. The purpose of our work is to understand how the NRF2/HO-1 axis, an important anti-inflammatory pathway, regulates IRF5 to find new molecular targets to counteract neuroinflammation. METHODS Human microglial cell line (HMC3) and murine macrophage-like cells (RAW264.7) were cultured under standard conditions and treated with 100 ng/ml Lipopolysaccharide (LPS), 5 µM Sulforaphane (SFN), 10 µM Tin mesoporphyrin IX (SnMP) and with 20 nM Resiquimod (R848). HO-1 silencing was performed by using a specific pool of oligonucleotides. Western Blot analysis was performed to detect proteins of interest in the whole cell lysates and in nuclear fractions. TNFα release in cell culture medium were detected by using ELISA assay. mRNA expression was measured by RT-qPCR analysis. Statistical analysis was performed by applying ANOVA and Tukey’s multiple comparison tests. RESULTS Microglial cells exposure to SNF, a NRF2 activator, prevented the induction and the release of TNFα and significantly reduced IRF5 nuclear localization in response to LPS stimulation. Notably, SFN reduced IRF5 expression in microglial cells exposed also to R848, used to activate IRF5. Moreover, we observed that TNFα gene expression was higher in cells silenced for HO-1 or exposed to HO-1 inhibitor (SnMP) and exposed to LPS compared to LPS alone. Also, preliminary results showed that HO-1 silencing promotes IRF5 nuclear localization. These findings were confirmed in the murine macrophage-like cell line RAW264.7. CONCLUSIONS To conclude, our study demonstrates that the NRF2/HO-1 pathway limits IRF5 activity and reduces microglia’s proinflammatory activation. These findings provide new insights into the regulation of neuroinflammation that could inform future therapeutic strategies.