Fig. 4: Identificating of BDNF as a antagonistic ligand of TLR4 in macrophages.

A Schematic illustration of the workflow integrating protein microarray screening, IP-MS analysis, and subcellular localization filtering to determine the potential receptor for BDNF. B Venn diagram showing TLR4 and CCR1 as the two membrane proteins with potential BDNF-binding capacity. C Co-immunoprecipitation analysis in 293 T cells transfected with or without CCR1 and BDNF plasmids, demonstrating no detectable interaction between CCR1 and BDNF. D 293 T cells were transfected with or without TLR4, MD2, and BDNF plasmids. Co-immunoprecipitation revealed an interaction between BDNF and TLR4, but not MD2. E Surface plasmon resonance analysis of direct affinity between BDNF and TLR4. (F-H) Primary macrophages were pre-treated with or without 5, 10 nM rBDNF for 1 h and then exposed to 500 ng/mL LPS for 0.5 h. F The binding level of BDNF with TLR4 in protein lysates was determined by ELISA assay. G TLR4 was immunoprecipitated (IP) and the associated MD2 level was detected using immunoblotting (IB). H The binding level of MD2 with TLR4 in protein lysates was determined by ELISA assay. I Primary macrophages were pre-treated with or without rBDNF for 1 h and then exposed to 500 ng/mL LPS for 0.5 h. Activation of the MyD88-dependent pathway was assessed by probing for IκB-α, phosphorylated TAK1, and phosphorylated MAPK (ERK, JNK, and p38) proteins. Activation of the TRIF-dependent pathway was assessed by probing for phosphorylated TBK1 and IRF3. Unphosphorylated proteins were used as the controls. J Wild-type macrophages transfected with luciferase reporter plasmids were pre-treated with rBDNF and exposed to LPS for 24 h. Relative luciferase activity of NF-κB p65, AP-1, and IRFs was measured. K Primary macrophages were pre-treated with rBDNF and then exposed to LPS for 6 h. Relative mRNA levels of pro-inflammatory factors Tnf, Il6, Il1b, Ccl2, Ifna1, Ifnb1, and Ifng were determined. L Primary macrophages were pre-treated with rBDNF and then exposed to LPS for 24 h. Cytokine levels of TNF-α, IL-6, and IFN-α in cultured medium were measured. M Schematic illustration of mice undergoing bone marrow transplantation, ALI modeling, and rBDNF injection. N Representative H&E-stained images of lung tissues. O Quantification of lung injury scores for (N). P Total protein levels in BALF samples. Q Lung wet/dry weight ratio. R Total cell counts in BALF samples. S Survival curve of born marrow transplanted mice with or without rBDNF injection in an E.coli-induced sepsis model. Data are represented as mean ± SEM by one-way ANOVA with Tukey’s test. n = 3 (H, J), or n = 4 (F, L, and K), or n = 6 (O–R) per group (E–G, J–M). Source data are provided as a Source Data file.