Effect Of Bcg On IL-35 Secretory Modulation By Macrophages

Objective: tuberculosis is still the leading cause of death of infectious diseases in the world.It is estimated that there are still 3 million cases of undiagnosed and untreated tuberculosis in 2013.Early disease,extrapulmonary tuberculosis,tuberculosis / human immunodeficiency virus(HIV)Co-infection,childhood tuberculosis and multidrug-resistant tuberculosis are particularly difficult to diagnose and treat.Autopsy studies from Africa have confirmed a large number of undiagnosed TB,subclinical TB and TB co morbidity with HIV,suppurative pneumonia and other infectious and non communicable diseases.This unacceptable state of affairs shows that current approaches to the diagnosis,treatment,management and prevention of tuberculosis are still inadequate and continuous efforts are needed to combat the disease.The combination of antigens on Mycobacterium tuberculosis and pattern recognition receptors of phagocytes can cause these cells to secrete many kinds of small molecular weight proteins,commonly known as cytokines.These cytokines mainly play an immune regulatory role through paracrine effect.Some cytokines can help the body to eliminate Mycobacterium tuberculosis,but there are also some cytokines that contribute to the survival of Mycobacterium tuberculosis.1.Interleukin-12(IL-12)family is a very important interleukin family.The unique feature of this family is that only heterodimer cytokines,including IL-12,IL-23,IL-27 and IL-35,have unique characteristics in immune response.IL-35 consists of EBI3 and p35 subunits IL-35.IL-35,a newly identified member of this family,is a potent inhibitory cytokine produced by regulatory T cells derived from thymus.IL-35 can induce the production of IL-10,inhibit TH2 polarization and the secretion of Th2-related cytokines,and inhibit TH17 polarization by decreasing the ratio of IL-23/IL-27.Macrophages are important innate immune cells in the Alveoli,whether macrophages secrete IL-35 and the effect of BCG on IL-35 secretion have not been reported.This study first observed the expression of IL-35 in patients with tuberculosis,focused on the effect of BCG(BCG)on IL-35 EBI3 subunit of macrophages,and further explored the regulatory mechanism of BCG on IL-35 EBI3.Methods:1.The level of IL-35 was detected in the serum of 25 tuberculosis patients and 25 healthy controls(from Ping an hospital and Hebei provincial chest hospital).2.Preparation and culture of bone marrow-derived macrophages from mice.3.The expression of mIL-35 EBI3 mRNA in RAW264.7 cells and BMDM was detected by real-time PCR.4.The expression of EBI3 protein in RAW264.7 cells was detected by Western blot.5.After RAW cells were pretreated with different inhibitors of transcription pathway,the effect of BCG on mIL-35 EBI3 mRNA expression was observed.6.Random primers were designed for IL-35 EBI3 DNA,and real-time quantitative PCR was performed to detect the pre-transcriptional changes of IL-35 EBI3 in RAW264.7 cells after stimulated by BCG.7.RAW264.7 cells were stimulated with BCG and then added with transcription inhibitor 5-methylbenzimidazole(diclofenzimidazole)+ actinomycin D(ACD)to prevent the production of new RNA.EBI3 mRNA decay was detected at each time point in the two groups.8.RAW264.7 cells were treated with different concentrations of p38 signal transduction pathway blocker SB203580,and then stimulated with BCG and then EBI3 mRNA levels were detected by real-time PCR.9.RAW264.7 cells were transfected with plasmid p38,and then stimulated by BCG,EBI3 mRNA levels were detected by real-time PCR.Results:1.The level of IL-35 in serum of TB patients was significantly higher than that in normal subjects.2.The expression of mIL-35 EBI3 mRNA and protein in RAW264.7 and BMDM were significantly increased by BCG stimulation.3.The results showed that BCG could not promote the expression of EBI3 at transcriptional level,but could reduce the decay rate of EBI3 mRNA.4.MAPK p38 pathway plays an important role in the high expression of EBI3 induced by BCG.5.The expression of EBI3 mRNA was significantly decreased after treatment with SB203580,a MAPK p38 pathway blocker.RAW264.7 cells were transfected with control plasmid and MAPK p38 plasmid respectively and stimulated with BCG.Real time quantitative PCR showed that EBI3 mRNA level of RAW264.7 cells with high expression of MAPK p38 was significantly increased after stimulation with BCG.Conclusions:1.The expression of IL-35 in serum of patients with tuberculosis was increased,expression of mIL-35 EBI3 mRNA and protein in macrophages were increased by BCG stimulation.2.BCG could not promote the transcription of IL-35 EBI3,but could inhibit the decay of IL-35 EBI3.3.MAPK p38 pathway plays an important role in the high expression of EBI3 induced by BCG.