Microglial TLR7 contributes to traumatic brain injury-induced neuroinflammation in mice

Neuroinflammation driven by activated microglia is an important component of secondary injury after traumatic brain injury (TBI), but the underlying molecular mechanisms remain incompletely understood. Here, we investigated the role of microglial Toll-like receptor 7 (TLR7) in TBI-induced neuroinflammation and evaluated the therapeutic effect of the TLR7-targeting compound Enpatoran (M5049). Analysis of public single-cell transcriptomic datasets showed that TLR7 is preferentially expressed in microglia among resident brain cells. In the controlled cortical impact (CCI) mouse TBI model, TBI increased TLR7 expression in microglia and elevated the abundance of cleaved-TLR7 in the peri-contusional cortex. Knockdown of the asparaginyl endopeptidase (AEP) attenuated TLR7 cleavage and downstream signaling in cultured microglia. Deletion of AEP decreased cleaved-TLR7 and inflammatory marker expression in TBI mice, indicating an AEP-mediated proteolytic processing of TLR7 and suggesting TLR7's involvement in TBI inflammation. Global TLR7 deletion in mice failed to reduce TBI-induced inflammation and was accompanied by marked TLR8 upregulation. In contrast, inducible microglia-specific TLR7 deletion in mice attenuated CD86, TNF-α, and IL-1β expression without a pronounced TLR8 upregulation. The TLR7 inhibitor M5049 suppressed TLR7 agonist-induced inflammation in cultured microglia without inhibiting mouse TLR8. In TBI mice, treatment with M5049 at 0.1, 0.3, and 3 mg/kg all significantly reduced TBI-induced IL-1β and IL-6 mRNA expression. Pharmacological tests further showed that M5049 is effective in suppressing TBI-induced microglial activation and inflammatory cytokine secretion in wild-type mice but lost this efficacy in TLR7 knockout mice. Therefore, M5049's anti-inflammatory action relies on the presence of TLR7. Additionally, M5049 was not able to rescue glutamate- and H₂O₂-induced cell death in HT22 mouse hippocampus neuronal cells, or broadly suppress the examined peripheral immune transcriptional program. These findings identify microglial TLR7 as an important player in TBI-induced neuroinflammation and support a predominantly TLR7-dependent anti-inflammatory effect of M5049.