Based on significantly increased TLR4, IκB, TNF-α, IL-1β protein expressions, and NF-κB nuclear protein expression induced by E. coli, we inferred that it activated TLR4/NF-κB signaling pathways in bMECs and macrophages, consistent with E. coli causing mastitis through the TLR4/NF-κB pathway (Ma et al., 2019). Here, TLR4 is linked to mastitis.