The Function Of De-ubiquitination Enzyme TNFAIP3in Brucella Intracellular Survival

Brucellosis is a zoonotic disease caused by a number of Brucella species.Brucella is an intracellular bacterial pathogen that causes abortion and infertility inmammals and leads to a debilitating febrile illness that can progress into a long lastingdisease with severe complications in humans. Its virulence depends on survival andreplication properties in host cells. The preferential reproductive niche of thisbacterium corresponds to the intracellular milieu of macrophages (M). At early timeof the infection, Brucella uses the stealthy and furtive strategies and stir upa lowactivation of the inflammatory reaction. The innate immune is suppressed, whichresults in releasing a little of TNF-α and IL-6and other inflammatory cytokines inhost cells. After this long incubation period, Brucella becomes evident, with theconcomitant establishment of the chronic infection and can replicate in themacrophages. Yet the underlying mechanisms and the nature of the replicativecompartment remain unclear. Therefore, it is very important to learn the Brucella howto evade supervision of the innate immune.De-ubiquitination enzyme TNFAIP3is also called zine finger protein A20, whichis a negative regulation protein in the TLRs and NLRs signaling pathway.Theprevious study showed that the A20mRNA level was increased at the early stage ofBrucella infection in macrophages. To determine whether A20is involved inregulation of Brucella-infected innate immunity, the J774A.1macrophageof A20expression and knock-down were constructed through lentivirus package’s method.Briefly, the recombinant lentivirus was packaged by co-tranfecting293T cells usingexpression plasmid, packaging plasmid and envelope plasmid. The expression ofprotein A20was detected by Western blotting. The expression of IκB-α was detectedin J774A.1cells after LPS treatment by Western blotting and the concentrations of TNF-α, IL-6in the supernatants were determined by cytokine ELISA kits. Theconcentrations of TNF-α in the supernatants was determined in J774A.1cells afterBrucella infection. Finally, infected cells were lysed with0.1%Triton X-100inphosphate-buffered saline (PBS) at the indicated times and serial dilutions of lysateswere rapidly plated onto tryptic soy agar plates to enumerate clone formation unint(CFU).Identification of restriction enzyme digestion and DNA sequencing showed thelentiviral vector was successfully constructed. The green fluorescent cells wereobserved under the fluorescence microscope after the recombinant lentivirusestransduced cells48h, which demonstrated the recombinant lentiviruses expressing andknocking-down A20gene were packaged successfully. Our data indicated that A20was expressed in J774A.1cells. The degradation IκB-α was abrogated in response toLPS stimulation in the A20over-expression J774A.1cells. The productions ofinflammatory cytokine TNF-α, IL-6were decreased significantly compared withnon-expression A20J774A.1cells. Thus, the macrophage J774A.1cell line model ofA20expression was constructed successfully．In the knock-down group, our datashowed that A20was suppressed. The degradation of IκB-α was not reversed inresponse to LPS stimulation in A20knock-down J774A.1cells. The productions ofinflammatory cytokine TNF-α, IL-6were increased significantly compared withnon-expression A20J774A.1cells. Thus, the macrophage J774A.1cell line model ofA20knock-down was successfully constructed.The production of inflammatory cytokine TNF-α was decreased dramatically inA20over-expression group upon Brucella post infection. However, in the A20knock-down J774A.1cells, the production of TNF-α was increased significantly.Compared with the control group, the number of intracellular bacteria (CFU) wasincreased in the A20over-expression J774A.1cells, but CFU was decreased in theA20knock-down J774A.1cells.Taken together, these results indicated that A20play a negative role in the regulation of Brucella-infected innate immunity, which provided experimental datafor future to investigate how A20implement the function of negative regulation inBrucella infection.