Giardia duodenalis is a protozoan parasite responsible for waterborne diarrheal diseases in humans and animals worldwide. TLR4 is a key pattern recognition receptor of the innate immune system, and MHC-II bridges innate and adaptive responses, while role of these receptors in the activation of macrophages by G. duodenalis still needs further investigation. This study confirmed knockdown efficiency using RT-qPCR and western blotting then employed multiple molecular and cellular assays to investigate the functions of TLR4 and MHC-II in RAW264.7 macrophages during defense against G. duodenalis. Results showed that the significant upregulation of proinflammatory cytokines (e.g., 5.18-fold increase in IL-6 expression) and marked activation of NF-κB pathways (marked enhancement of p65 fluorescence in the cell nucleus) in macrophages following G. duodenalis infection were dependent on TLR4, as demonstrated by their attenuation after TLR4 knockdown. Interestingly, we also observed that the knockdown of MHC-II produced similar results, and the elevated expression of MHC-II in macrophages induced by G. duodenalis was inhibited by the interference of class II transactivating factor (CIITA). Overall, CIITA/MHC-II/NF-κB pathway is critical for full activation of TLR4-mediated innate immune response in macrophage initiated by G. duodenalis, which elaborated the innate immune network in the interaction between G. duodenalis and macrophages, holds significant implications for the immunotherapeutic strategies targeting Giardiasis.

To Cite This Article: Song P, Li L, Du J, Zuo S, Dong H, Li J, Wu Y, Zhang L, Liu F and Dai H, 2025. The CIITA/MHC-II/NF-κB Pathway Mediates Full Activation of TLR4-Driven Innate Immunity in RAW264.7 Macrophages Challenged with Giardia duodenalis. Pak Vet J. http://dx.doi.org/10.29261/pakvetj/2025.322