Research On The Mechanism Of TLR4 In Ricin Toxin-induced Inflammatory Injury In Macrophages

Ricin toxin(RT)is a natural plant-derived protein toxin from the seed of castor beans that belongs to a family of type II ribosome-inactivating proteins(RIPs).It has extremely strong cytotoxicity to all mammalian eukaryotic cells.RT is a highly glycosylated dimer protein,which is composed of RTA and RTB chains covalently linked by disulfide bonds.Among them,RTA has N-glycosidase activity,which can hydrolyze ribosomes and inhibit protein synthesis.RTB has lectin activity,which can bind to eukaryotic surface glycoproteins and glycolipids to help RTA enter into cells and exert toxicity.The toxicity of RT is dose-dependent.The toxicity of high-dose RT is mainly characterized by inhibition of protein synthesis.Low-dose RT can induce apoptosis,membrane damage,membrane structure and function changes and cell inflammatory injury.In addition,according to the way in which the body is exposed to RT,its toxicity is also different.RT is the most toxic to enter the body through inhalation,which can lead to severe lung injury.After entering the lungs,RT binds to the mannose receptor on the surface of macrophages,thus increasing its migration rate.Macrophages play a crucial role in innate immunity and the adaptive immune response as the first line of host defense against bacterial infections and various types of invading pathogens.Upon activation,macrophages release types of cytokines to remove pathogens.TLRs,important part of innate immune response highly expressed by macrophages,which can promote the expansion of antigen-presenting cells and the synthesis and secretion of proinflammatory cytokines that induce inflammation,are crucial bridges connecting innate and adaptive immunity.TLR4 signaling pathways play an important role in the activation of immune cells,and its mediating cellular inflammatory response signaling pathways are mainly My D88-dependent pathway and TRIF-dependent pathway.However,there are few reports about the effect of RT on innate immune response and its mechanism.Here,RT was used as the research object to explore the key role of TLR4 in RT-induced macrophage inflammatory injury by using molecular biology and CRISPR/Cas9 gene editing techniques.It is of great significance to clarify the mechanism of RT-induced macrophage inflammatory injury,to find a new way to treat RT poisoning and to develop anti-RT drugs or vaccines.In this study,MTT was used to detect the effect of RT on the cell viability of mouse mononuclear macrophage line RAW264.7 cells and human mononuclear macrophage line THP-1 cells in vitro.Results showed that the IC50 of RAW264.7 cells treated by RT was about 36.14 ng/m L,and the IC50 of THP-1 cells was 8.13 ng/m L.Secondly,LPS(TLR4 activators),PGN(TLR2 activators),Poly I:C(TLR3 activators)and RT were used to stimulate the cells.ELISA assays were used to detect the effect the expression level of inflammatory factors in macrophages.Results showed that RT treatment promoted a significant increase in TNF-α and IL-6 secretion induced by LPS and PGN,indicating that RT treatment induced TNF-α and IL-6 production may through the My D88-dependent pathway.Moreover,RT promoted a significant increase in LPS-and Poly I:C-induced IFN-β secretion levels,indicating that RT induces IFN-β secretion may through the TRIF-dependent pathway.IκB-α and IRF3 are key transcription factors in My D88-dependent and TRIF-dependent pathways,respectively.Then,by using Western Blot,the activation of TLR4-mediated signaling pathway by RT was studied by detecting the protein expression of TLR4,My D88,TRIF,P-IκB-α,and P-IRF3.Results showed that RT treatment up-regulated TLR4,My D88,and TRIF protein expression levels,up-regulated LPS-,PGN-,and Poly I:C-induced increases in P-IκB-α protein expression,and up-regulated LPS-and Poly I:C-induced IRF3 protein expression.Overall,these results confirmed that RT can activate the My D88-dependent pathway and the TRIF-dependent pathway in the TLR4 signaling pathways.Because of the inflammatory response mediated by TLR4 mainly through My D88 and TRIF-dependent pathways.With the aim to further confirm that RT can induce inflammation through that two signaling pathways,a RAW264.7 cell line stably knockout of My D88/TRIF gene was established by CRISPR/Cas9.The secretion levels of TNF-α and IFN-β were significantly decreased in RAW264.7 cells stably knockout of My D88/TRIF gene,which confirmed that RT induced cellular inflammatory injury through My D88 and TRIF-dependent pathways.Finally,in order to verify the protein-protein interaction between RT and TLR4,the binding of RT and TLR4 was detected by Co-immunoprecipitation.Results showed that there was protein-protein interaction between RT and TLR4.Therefore,TLR4 is a receptor of RT,and TLR4-RT binding activates the inflammatory signaling pathway.In summary,this study found for the first time that TLR4 mediates cellular inflammatory response as a receptor of RT,confirming that RT can activate TLR4 and its downstream My D88-dependent and TRIF-dependent pathways to mediate cellular inflammatory response,and reveal the possible mechanism of low-dose RT activating TLR4 signaling pathways,which will help to identify key target molecules in the treatment of RT-induced inflammatory injury and improve the therapeutic efficiency of RT poisoning.It can lay a theoretical foundation for the development of anti-RT drugs or vaccines.In addition,the inflammatory response of macrophages activated by RT may also be caused by other receptors or signaling pathways,these unknown details need to be further explored.