Different doses of systemic LPS induce different degrees of polarization of microglia and astrocytes

Abstract

Background Microglia and astrocytes are activated in different phenotypes to exert opposite effects. The recently reported intraperitoneal injection of 5 mg/kg lipopolysaccharides (LPS) to promote A1 astrocytes by activating M1 microglia was found to cause high mortality. Furthermore, reported doses of systemic LPS used to induce M1 microglia vary widely (0.1 ~ 5 mg/kg). We aimed to study microglia and astrocytes polarization induced by various LPS doses in the central nervous system, and assess whether downregulation of C3a receptor (C3aR) in astrocytes contributes to an increased A2/A1 ratio. Methods Rats were randomly divided into six LPS dosage groups (0, 0.1, 0.33, 1, 3, and 5 mg/kg, intraperitoneally). Seventy-two genes for A1, A2, A-pan, M1, and M2 markers were detected by real-time polymerase chain reaction 24 hours after LPS treatment in the cerebral cortex, hippocampus, and spinal cord. C3aR in astrocytes was knocked down by intrathecal injection of AAV-C3aR-GFAP 21 days before LPS administration. Co-immunofluorescence of C3aR with microglia, astrocytes, and neuron markers were performed to verify the specificity of C3aR knockdown in astrocytes. Changes in the 72 genes in the spinal cord were detected again 24 hours after LPS injection. Results Systemic LPS activated not only A1 and M1, but also A2, M2, and A-pan in the cerebral cortex, hippocampus, and spinal cord. The same LPS dose induced a similar activation level of M1 and M2, both of which were upregulated with increasing LPS. A1 and A-pan were polarized more than A2 at all LPS doses in the cortex and spinal cord. Microglia were more activated at 5 mg/kg than at 3 mg/kg LPS, but astrocytes presented no activation advantage at 5 mg/kg LPS. Marco, Ym1, and C3 showed a significant dose-dependent increase in LPS concentration. Specific knockdown of C3aR in astrocytes upregulated more markers of A2 than A1 and A-pan. Conclusions A larger systemic LPS dose contributes to greater polarization of M1 and M2 microglia, but no dominant phenotype. More A1 and A-pan astrocytes were activated than A2, even at low LPS doses. Downregulation of C3aR in astrocytes contributes to the polarization of anti-inflammatory phenotypes induced by LPS.