| Research Background and ObjectivesTuberculosis(TB)caused by Mycobacterium tuberculosis(MTB)is a chronic infectious disease that seriously harms human health.The high incidence of tuberculosis and the continuous emergence of drug-resistant strains are currently the thorny problems.Problems,therefore,urgently need to seek new and effective anti-MTB therapy,and immunotherapy is a better choice.Dendritic cells(DCs)are important bridge cells that link the body’s innate immunity and adaptive immunity.They are also known as the most powerful antigen presenting cells.Therefore,activation of DCs can both enhance innate immunity and adaptive immunity.There are multiple pattern recognition receptors on DCs,of which TLR4 and NOD2 are pattern recognition receptors on the cell membrane and intracellular,respectively,which play an important role in infection.TLR4(Toll like receptor 4)is distributed on the cell membrane and belongs to the transmembrane receptor.LPS is the specific activation ligand of TLR4.NOD2(nucleotide-binding oligomerization domain 2)is mainly distributed in the cytoplasm of cells,and is an intracellular recognition receptor,and muramyl dipeptide(MDP)is its specific activating ligand.DC stimulates NF-κB to express inflammatory cytokines such as IL-6,IL-1β and TNF-α under the stimulation of ligands.Therefore,IL-6 is one of the important cytokines for DC activation.In this study,we aimed to investigate whether TRL4-NOD2 coordinated signal transduction can enhance the activity of DC,and whether activated DC can inhibit the growth of MTB,and provide a new direction and theoretical basis for the discovery of novel anti-infection immunotherapy.MethodsMouse DC2.4 cells were cultured and DC2.4 was stimulated with TLR4 ligand(LPS),NOD2 ligand(MDP)and T4N2 dual ligand(TLR4-NOD2 Ligand,T4N2L)respectively.Enzyme-linked immunosorbent assay(ELISA)the IL-6 concentration in the cell supernatant was measured at different time points.Then by constructing a lentiviral vector for silencing the NOD2 gene in mouse DC2.4 cells,after transfection of mouse DC2.4,verification of successful silencing of NOD2 by RT-PCR.After silence was successful,DC2.4 was stimulated with different ligands.The concentration of IL-6 in the supernatant after 24 h was tested by enzyme-linked immunosorbent assay(ELISA)to verify the synergistic effect.After infecting DCs with MTB,DCs were stimulated with different ligands.After 24 hours,the cells were lysed and MTBs were collected,inoculated in L-J medium,and the colonies were counted after 3 weeks.Experimental data were analyzed using SPSS17.0 statistical software.Results1.Compared with blank group,LPS group,MDP group,the concentration of cytokine IL-6 began to increase from 6h,and were higher than the blank control group,the difference was statistically significant(P<0.05),and T4N2 L group The concentration of cytokine IL-6 was significantly higher than that of the single ligand stimulation group and the blank control group,and the difference was statistically significant(P<0.05).2.After silence NOD2 gene,the concentration of IL-6 in MDP group was significantly lower than that before silencing,while the concentration of IL-6 in T4N2 double-ligand stimulation group was significantly lower than that before silencing(P<0.05)3.DCs of different ligand stimulation groups had inhibitory effects on the growth of MTB,and DCs of T4N2 L double ligand stimulation group could significantly inhibit the growth of MTB.Conclusion1.Single ligand stimulation can enhance DC activation.2.The signal transduction of TLR4-NOD2 can significantly enhance the activation of DC.3.After the silencing of NOD2 gene,the activation of DCs was significantly decreased,indicating that TLR4-NOD2 signal transduction can significantly enhance the activation of DCs,and the combined action can significantly increase the activation of dendritic cells.4.Activated DCs stimulated by TLR4-NOD2 cooperative signal can significantly inhibit the growth of MTB. |
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