Brucella Omp25 Protein Regulates IL-12 Expression Via PD-1 Induced MicroRNA

Brucellosis is a zoonotic disease caused by Brucella infection.The clinical manifestation is various,animal infection leads to abortion of pregnant female animals,orchitis in male animals;human infection can cause symptoms such as wave heat,excessive sweating,joint swelling and pain,hepatomegaly and splenomegaly.It not only causes significant economic losses to the livestock industry and also a serious threat to human health.Brucella has a variety of pathogenic factors,and employs a variety of mechanisms to escape body's immunity,allowing them to survive and replicate in the cells.As a kind of effector cells of innate immunity,macrophages play an important role in resisting pathogen invasion.However,Brucella can escape the killing effect of macrophages and survive and multiply in macrophages,causing chronic infection of the body.Studies have shown that wild-type Brucella infection inhibits the secretion of TNF-? and IL-12 in macrophages,and also inhibits macrophage phagocytosis and antigen presentation,thereby inhibiting Th1 cell immunity,resulting in the body's inability to effectively eliminate Brucella bacteria.However,compared with the wild type,the B.suis Omp25 deletion mutant has a weaker inhibitory effect on macrophage secretion of TNF-? and IL-12.Previous studies have confirmed that Omp25 can inhibit the secretion of TNF-? in macrophages.However,whether Omp25 protein can exert immune escape by regulation the IL-12 secretion of macrophages has not been reported.In this study,THP-1 cells were infected with Omp25 recombinant adenovirus rAd-Omp25 and stimulated by LPS/R848,the effect of Omp25 on the production of IL-12p70 in macrophages was detected by ELISA,and then the effects of Omp25 on IL-12p35 and p40 were detected at the transcriptional and post-transcriptional levels.The miRNAs and signaling pathways involved in the regulation of IL-12 production were identified by dual luciferase assay,Q-PCR and Western Blot.Finally,the regulation and mechanism of Omp25 protein on the production of IL-12 in macrophage were clarified,which provide a theoretical basis for further elucidation of the immune escape mechanism of Brucella.The study obtained the following results:1.THP-1 cells infected with Omp25 recombinant adenovirus stably expressed Omp25 in cells.ELISA showed that Omp25 inhibited the production of IL-12p70 induced by LPS/R848.ELISA and Western Blot results showed that Omp25 inhibited the expression of IL-12p35 and p40 induced by LPS/R848.Q-PCR analysis showed that Omp25 did not affect the mRNA level of il12 A,but significantly down-regulated the mRNA level of il12 B.By detecting the promoter activity of il12A(p35)and il12B(p40),we found that Omp25 inhibited the transcription of il12B(p40),but had no significant effect on the transcription of il12A(p35).By detecting the transcriptional activity of NF-?B p65 and the level of I?B and I?B phosphorylation,we found that Omp25 inhibited the activity of NF-?B p65 signaling pathway through inhibiting I?B phosphorylation and NF-?B p65 entry into the nucleus.2.Q-PCR results showed that Omp25 could up-regulate miR-23 b,miR-21-5p and miR-155;ELISA and Western Blot assays showed that miR-23 b and miR-155 were correlated with the inhibitory effect of Omp25 on the expression of IL-12p40,miR-21-5p was correlated with the inhibitory effect of Omp25 on the expression of IL-12p35;The dual luciferase reporter assay further confirmed that miR-21-5p and miR-23 b regulate the expression of IL-12p35 and IL-12p40 at the post-transcriptional level by targeting the 3'UTR of il12 A and il12 B,respectively.Western Blot assay showed that Omp25-induced miR-155 inhibited the transcription of il12 B by targeting TAB2;Flow cytometry and ELISA showed that Omp25 inhibited the expression of IL-12 by up-regulating PD-1.Q-PCR results showed that blocking PD-1 inhibited the expression of miR-23 b,miR-21-5p and miR-155 induced by Omp25,indicating that Omp25 regulates the expression of IL-12 in human monocytes/macrophages mainly through three miRNAs induced by PD-1.In summary,this study used Omp25 recombinant adenovirus to infect mononuclear cell THP-1 and stably expressed Omp25 protein.Results have shown that Omp25 protein inhibits the production of IL-12 induced by LPS/R848.Omp25 upregulates miR-155,miR-23 b and miR-21-5p in human monocytes/macrophages.miR-21-5p and miR-23 b regulate the expression of IL-12p35 and IL-12p40 at the post-transcriptional level by targeting the 3'UTR of il12 A and il12 B,respectively.miR-155 inhibit NF-?B signaling pathway by targeting TAB2 and inhibits transcription of il12 B at the transcriptional level;Omp25 upregulates miR-23 b,miR-21-5p and miR-155 by activating PD-1 signaling,thereby inhibiting the expression of IL-12 induced by LPS/R848.