Low-dose TLR9 agonist ODN 2395 drives microglial neuroprotective activation by CREB phosphorylation

Background: The Toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine (CpG) oligodeoxynucleotide (ODN) has been reported to induce microglial pro-inflammatory polarization, contributing to neuronal damage. This study investigates the anti-inflammatory activation of microglia by low-dose CpG ODN 2395 and its underlying mechanism. Methods and Results: BV2 microglia, primary mouse mixed glial cultures, and primary mouse mesencephalic neuron-glial cultures were treated with ODN 2395. RT-qPCR, ELISA, Western blotting, and immunocytochemistry were employed. Lipopolysaccharide (LPS) was used to induce neurotoxicity and serve as a control to compare inflammatory responses. Low-dose ODN 2395 (100–200 nM) causes minimal mitochondrial ROS production and promotes anti-inflammatory polarization in microglia, characterized by high levels of Arginase 1 (Arg1), IL-10, and brain-derived neurotrophic factor (BDNF), but low expression of iNOS, TNF-α, and IL-1β. Phosphorylated cAMP response element-binding protein (CREB) is essential for the induction of anti-inflammatory and neuroprotective factors by ODN 2395. Even with endoplasmic reticulum stress, a common pathology in neuroinflammatory disorders, low-dose ODN 2395-activated microglia hardly release mature IL-1β, partially due to defective NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) priming. NLRP3 knockout validated its crucial role in microglial mature IL-1β release. In neuron-glial cultures, low-dose ODN 2395 significantly mitigated LPS-induced neurotoxicity, evidenced by preserved neurons and reduced reactive microgliosis. Conclusions: Our findings provide novel insights into the immune responses of microglial TLR9 activation and suggest stimulatory intensity of TLR9 as a potential determinant for the immunotherapeutic strategy.