Study On The Regulation Of Interleukin-10 Expression And Gene Polymorphisms Of Monocyte Chemoattractant Protein-1 In Tuberculosis

ObjectiveTuberculosis(TB)is a chronic infectious disease transmitted by the Mycobacterium tuberculosis(MTB)through the respiratory tract.Mycobacterium tuberculosis can invade and infect many human organs and lung is the most common one.Therefore,pulmonary tuberculosis is the most common form of tuberculosis.Tuberculosis is an ancient disease that endanger the health and safety of human life for a long time.At present,tuberculosis is the ninth largest cause of human death in the world and the main cause of human death caused by a single pathogen infection.In the 2017 global tuberculosis report,WHO estimated that there were 10.4 million people fell ill with TB in 2016:90%were adults,65%were male,10%were people living with HIV and 56%were in five countries:India,Indonesia,China,the Philippines and Pakistan.So,the global burden of tuberculosis is still very heavy,which is the most serious in Africa and Asia.China is one of the countries with a high burden of tuberculosis,and the situation of tuberculosis prevention and related research is urgent.Human body can immediately start up a series of immune action to respond to the invasion of Mycobacterium tuberculosis.Macrophages participate in the innate and acquired immune response against Mycobacterium tuberculosis.Cytokines such as interleukin-10(IL-10)and chemokines such as monocyte chemoattractant protein-1(MCP-1),secreted by macrophages,can directly affect the immune ability againsttuberculosis and thereby alter the final results of such infection.Studying the immune related factors against tuberculosis infection is helpful for us to understand the infection mechanism of tuberculosis in depth and find new ideas about the prevention,control and treatment of tuberculosis.IL-10 is an inhibitory cytokine,which is closely related to the immune escape ofMycobacterium tuberculosis,the latent tuberculosis infection(LTBI)and the recurrence of tuberculosis.The regulation mechanism of the expression of IL-10 in tuberculosis infection has not been fully elucidated.Histone deacetylases(HDACs)is a key enzyme family which can maintain the acetylation balance of nucleosomes in chromosomes.HDAC6 and HD AC 11 are two important members of HDACs.It has been found that HDAC6 and HD AC 11 can regulate IL-10 expression in antigen presenting cells(APCs)stimulated by lipopolysaccharide in vitro.There are no any reports about the regulation of IL-10 expression through HDAC6 and HD AC 11 in tuberculosis infection so far.Thus,in the first part of our research,we preliminarily discussed this question.MCP-1 is an important chemokine.It can help monocytes and activated T lymphocytes to recruit to the infection sites and promote the formation of tuberculous granuloma.It is closely related to the progress or prognosis of tuberculosis.Studies have found that the gene polymorphism of rs 1024611,which locates the 5 'end of human MCP-1 transcription start sites,may be related to the susceptibility of tuberculosis.While,such correlation in studies about different regions or human goups is different.Shandong province has become the second largest province in China with the total population over one hundred million.The epidemic situation of tuberculosis in Shandong can not be ignored.Therefore,it is necessary to explore the relationship between the gene polymorphism of MCP-1 rs 1024611 in Shandong and the susceptibility to tuberculosis.In the second part of our research,we analyzed and discussed the relationship between MCP-1 gene polymorphism and susceptibility to active pulmonary tuberculosis in Shandong Han population.Therefore,in this paper,tuberculosis is the disease that we are interested in.We put our study focus on the macrophages which play an important role in the process of humane immune reaction against tuberculosis infection.We took IL-10 and MCP-1,the two important protein expressed and secreated by macrophages,as our research goals.Through the cell model of tuberculosis infection in vitro and case-control study,we explored respectively the regulation mechanism of IL-10 expression and the association of MCP-1 polymorphisms with susceptibility to pulmonary tuberculosis.We hope that our finding can provide further theoretical support and new ideas for the prevention and treatment of tuberculosis.MethodsSection one:Section one:Firstly,THP-1 cells were stimulated with PMA and then differentiaed into macrophages.These macrophages were then infected respectively with virulent MTB strain H37Rv using a series of multiplicity of infection(MOI)at different time.The expression level of IL-10 was measured and evaluated by ELISA and qRT-PCR and the optimum infection conditions were choosen.The cell model of tuberculosis infection in vitro was established;Secondly,by qRT-PCR and Western blot,we investigated the expression of HDAC6 and HD AC 11 respectively at an MOI of 10;Finally,macrophages,with the silenced gene expression of HDAC6 and HDAC11 by siRNA,were infected with H37Rv at an MOI of 10 and 24h.Then,the expression level of IL-10 was evaluated by ELISA and qRT-PCR.Section two:Firstly,unrelated Shandong Han Chinese adults were choosen as the study populations and were divided into active PTB(cases)and healthy adults(controls).All study populations were choosen with strict standard by CT examination,bacteriological examination,clinical features and T-SPOT.TB.Secondly,genomic DNA was extracted using peripheral white blood cells derived from patients with PTB and healthy controls.The MCP-1 rs1024611(AG)SNP genotyping was carried out then by real time fluorescence quantitative PCR;Finally,the association of MCP-1rs 1024611(AG)SNP genotype with PTB susceptibility was analysed between cases and controls.ResultsSection one:?IL-10 is upregulated in macrophages challenged with MTB in a dose and time-dependent manner.Our study confirms that macrophages are an important cellular source of IL-10.It was found that after infection of MTB at MOI of 10,IL-10 was dramatically increased and at 24 h,the expression of IL-10 reached the highest level.Thus,the cell model of tuberculosis infection in vitro was established.?MTB infection greatly altered the expression of HDAC6 and HD AC 11 in macrophages not only from the mRNA level but also from the protein level.We observed upregulation of HDAC6 levels and downregulation of HD AC 11 levels after MTB infection.We therefore hypothesized that MTB infection could promote the expression and secreation of IL-10 by disturbing the expression balance of HDAC6 and HDAC11.?HDAC6 knockdown cells by siRNA showed a significantly lower IL-10 expression both in protein and mRNA levels.?HDAC11 knockdown cells by siRNA showed a significantly higher IL-10 expression both in protein and mRNA levels.Section two:?A total of 183 patients with active PTB and 210 healthy adults were genotyped for MCP-1 rs1024611.Patient age was 42.2 ± 20.1 years(mean ± SD)and sex ratio was 125/58 = 2.16(M/F).Healthy adults recruited as controls were largely age-(42.8 ± 14.1)and sex-matched(140/70 = 2.0).Both cases and controls belonged to the Han Chinese ethnic group living in the Shandong area(East China).?HardyWeinberg equilibrium of the genotype distribution among the cases and controls was tested by a x2 test.The P values of the two groups were all greater than 0.05,indicating that the selected subjects of the two groups had reached the Hardy-Weinberg genetic balance and the research subjects were representative of the Shandong group.?Among 210 controls,97 had GG(46.2%),82 had GA(39.0%),and 31 had AA(14.8%)genotype,while there were 66 GG(36.1%),90GA(49.2%),and 27 AA(14.7%)genotypes among 183 cases.Clearly,the GG genotype was found at a higher frequency in healthy controls than in patients with PTB.The GA genotype exhibited signifcantly reduced risk of PTB(GG vs GA:OR,0.620;95%CI,0.427-1.428,P = 0.03).There was no difference in the AA genotype alone between cases and controls,however,when GA and AA genotypes were grouped together,the GG variant was exhibited at a signifcantly higher susceptibility to PTB(GG vs GA ?AA:OR,0.657;95%CI,0.438-0.986;P = 0.042).The A allele tended to have a lower PTB risk than the G allele did(OR:0.804,95%CI:0.601-1.076);however,the difference was not statistically signifcant(P = 0.142).?The GG genotype was observed at a lower frequency in male cases(35.2%)than in male controls(47.9%)and the risk was at a borderline signifcance as compared to the GA genotype(GG vs GA:OR,0.604;95%CI=:0.358-.019;P = 0.058).The cases with combined GA and AA genotypes exhibited significantly decreased PTB risk as com,pared to that exhibited by the GG variant carriers(GG vs GA + AA:OR,0.592;95%CI ?0.361-0.971;P = 0.037).In contrast,differences in the genotype distribution were not signifcant between female controls and cases(controls vs cases in female,P>0.05 in all the comparisons.Conclusion1.The results in vitro showed that when macrophages were infected with Mycobacterium tuberculosis,HDAC6 and HD AC 11 were closely related to the expression and secretion of IL-10 in macrophages.HDAC6 can upregulate the expression of IL-10 in infected macrophages,while HD AC 11 downregulates the expression of IL-10.Therefore,we determine that HDAC6 and HD AC 11 may be a new group of regulating factor for the expression of IL-10 produced by human macrophages in the process of human tuberculosis infection.This conclusion is the first report in the field of tuberculosis research.2.The results of case-control study and SNP genotyping showed that MCP-1 rs1024611 SNP genotype may be correlated with the susceptibility of active pulmonary tuberculosis in Han Chinese population of Shandong.Compared to GG genotype,GA genotype could significantly reduce the risk of active pulmonary tuberculosis.When GA and AA genotypes were grouped together,the GG variant was exhibited at a signifcantly higher susceptibility to active pulmonary tuberculosis.For the first time we find that when GA and AA genotypes were grouped together in male group,the GG variant was exhibited at a signifcantly higher susceptibility to active pulmonary tuberculosis.In contrast,differences in the genotype distribution were not signifcant between female controls and cases.