TLR4 and mastitis: However, LPS released by E. coli in vivo can not only activates TLR4 receptors on the cell membrane, but also directly enters into the cell to activate GSDMD, depending on CD14 (Figure S22A, Supporting Information).[22] Therefore, to further validate our in vitro results, we used TLR4−/− and GSDMD−/− mice to construct a mastitis model for in vivo validation, and found that both TLR4−/− and GSDMD−/− mice effectively alleviated the severity of mastitis.