The Role Of MicroRNA-146a In The Mycobacterium Tuberculosis Infection And Its Potential Mechanisms

Tuberculosis (TB) is an infectious disease caused by the bacillus Mycobacterium tuberculosis(M.tuberculosis). According to WHO’s report, there are20million active pulmonary TB patients worldwide,8million new cases of tuberculosis in the world annually and causing3million deaths while one third of the world population are infected. It is a major contagious disease for the death of adults. Currently, due to the epidemic of Multidrug-resistant TB (MDR-TB), extensively drug-resistant TB (XDR-TB) and TB/HIV co-infection, the situation of TB control and prevention seems more severe. Therefore, it is urgent that intensive study about infection and pathogenesis of TB should be taken.M.tuberculosis is a facultative intracellular pathogen which invades macrophage within a variety of pattern recognition receptors(PRRs) expressed on macrophages such as CR, MR, TLRs and NLRs. As a kind of important antigen presentation cells and innate immune effector cells, activated macrophages are the first barrier protecting our bodies from M. tuberculosis infection. Meanwhile, macrophages are the primary targets for intracellular invasion by M. tuberculosis and provide an essential niche to establish infection in the host. After internalized, M. tuberculosis are cleared by macrophages or escaped by a series of evasion mechanisms. The outcome of infection depends on bacteria virulence, the state of macrophages and interaction between macrophages and M.tuberculosis. Investigating the interaction between both sides would help us understand the early clearance of bacteria by hosts.microRNAs (miRNAs) belonged to a class of newly small non-coding RNAs, approximately20-24nucleotides long which bind to the3’-UTRs of the target mRNAs and leading degradation or transcriptional repression of target mRNA at post-transcriptional level. Since the first miRNA reported in1993, thousands of miRNAs were found in mammals though physiological role of majority of them remain unknown. In general, miRNAs act as key regulators in development, differentiation, homeostasis and cancers. Recent studies indicate that miRNA control has emerged as a critical regulatory principle in autoimmune diseases and cancer. The function of miRNA in M. tuberculosis infection, however, remains unclear.miR-146a are thought to be the first miRNA that can regulate innate immune system, many studies show that miR-146a take part in the process of inflammation and autoimmune diseases. A study indicates that miR-146a polymorphism is associated with increased risk of acquiring tuberculosis, yet no evidence disclosed the function of miR-146a in M.tuberculosis infection.Therefore, in this study we established infection models both in vivo and in vitro and analyzed the expression of miR-146a in M.tuberculosis infected macrophages. Then we explor the role of miR-146a in M.tuberculosis infection and its possible mechanisms. Our study would provide new theoretic base for study the immune mechanism of M. tuberculosis.Part I The expression of miR-146a after M. bovis BCG infectionFirst we detected the expression change of miR-146a in macrophages in response to M.bovis BCG infection. We established the mouse model of M.bovis BCG infection, pulmonary alveolar macrophage were harvested at day3,7,14,21,28and miR-146a expression change was analyzed using real-time RT-PCR. Also, murine macrophage cell line RAW264.7were infected with M.bovis BCG and the expression of miR-146a were detected at6h,12h,24h and36h by quantitative RT-PCR. The results showed that the expression of miR-146a was up-regulated6h after infection, reached its highest levels by24h and slowly decreased by36h. Then we infected macrophages with M. bovis BCG by different MOI. The result suggested that miR-146a expression was in dose-dependent manner. In addition to its up-regulation in RAW264.7cells, miR-146a expression was also up-regulated in mouse primary peritoneal macrophages and BMDMs. Above results indicated that M.bovis BCG can stimulate the expression of miR-146a both in vivo and in vitro, and this elevation is time-dependent and dose-dependent. The chemical structure of mycobacteria included a pedtidoglycan and glycolipid complex, this composition may play as antigens and stimulate macrophage as well. So we use live BCG and heat-killed BCG to infect or stimulate macrophages respectively. The results showed that both live or heat-killed BCG could up-regulated the expression of miR-146a, however, the level was not the same. Compared with heat-killed BCG, live BCG could induce obvious higher level of miR-146a.The result indicated that secretory protein produced by live bacteria may play a role in induction of miR-146a. Taken together these results we could draw the conclusion that M.bovis BCG infection could elevate the expression of miR-146a and miR-146a may play a regulatory role in the infection,To assess the signal pathway involved in regulating miR-146a expression following BCG infection, we pretreated RAW264.7cells with PDTC, SP600125, SB203580or U0126respectively, followed by exposure to BCG for24h. The result showed that only NF-kB inhibitor (PDTC) significantly suppressed the miR-146a expression. This result indicated that NF-kB is required for the induction of miR-146a in BCG infection.Part II The role and potential mechanisms underlying miR-146a participating in the infection of M.bovis BCGOur previous data have shown that the expression of miR-146a was notably increased after bacteria infection. Then next we want to find out the role of miR-146a in M.bovis BCG infection and its underlying mechanisms. RAW264.7cells were transiently transfected with miR-146a mimics or miR-146a inhibitor to overexpress or knockdown the endogenous level of this miRNA. Cells transfected with miR-146a mimics or miR-146a inhibitor were then analyzed for BCG-induced production of critical pro-inflammatory cytokines, TNF-α,IL-1β, IL-6and chemokine MCP-1at mRNA level using qRT-PCR and protein level by ELISA assay. As a result, miR-146a mimics induced a significant inhibition in both mRNA and protein level of TNF-α,IL-1β, IL-6and MCP-1after BCG infection compared to the control. miR-146a inhibitor, to the contrary, promoted the secretion of inflammatory cytokines in macrophages in response to infection. All results above indicated a strong suppressive role of miR-146a in M.bovis BCG-induced macrophage inflammatory response. After activated by bacteria, macrophage can killing intracellular pathgen via nitric oxide (NO) pathway. So we detected the NO production after BCG infection.. After transfected with miR-146a mimics, the NO release in the culture supernatant is remarkably decreased. On the contrary, macrophage transfected with miR-146a inhibitor showed great amount of NO release. Taken together, miR-146a may impair macrophage intracellular killing function via repression of NO releaseNext we checked the phagocytosis function of macrophage. Macrophages were transfected with miR-146a mimics, miR-146a inhibitor and negative control respectively. After48h, co-incubated them with FITC-labeled BCG for4h at MOI20:1, the percentage of cells that bound FITC-conjugated bacteria was measured by flow cytometry. The result demonstrated that overexpression of miR-146a can obviously promote the phagocytosis function while knockdown miR-146a has a negative effect on bacteria internalization.At early stage of infection, inflammatory response is very important for the bacteria clearance. Since we have understood that miR-146a could negative regulate inflammatory response to BCG infection, we examine whether miR-146a could affect the bacteria replication. Colony counting demonstrated that miR-146a mimics could promote BCG replication in macrophage. However, miR-146a showed only slight inhibition of bacteria proliferation. This maybe because of the function of inhibitor weakened after long time.PART Ⅲ miR-146a played a negative regulate role via directly targeting IRAK-1and TRAF-6in macrophagesAs previous mentioned, miRNAs played a regulatory role via complementary binding to the3’-UTR of target gene mRNA. So next we search the potential target or targets through documents and database. Among the target genes predicted by algorithm of Target Scan and miRanda, IRAK-1and TRAF-6were chosen for further study for their reported important role in macrophage inflammatory response,In order to detect whether miR-146a regulates IRAK-1and TRAF-6through direct3’UTR interaction, the3’UTR of IRAK-1and TRAF-6were cloned into a reporter plasmid downstream from luciferase and reporter assays were performed in RAW264.7cells. As a result, miR-146a obviously repressed the activity of luciferase fused to both IRAK-1and TRAF-63’UTR, which indicated a direct interaction between miR-146a and IRAK-1and TRAF-6in macrophages. To assess whether miR-146a had a functional role in the down-regulation of endogenous IRAK-1and TRAF-6expression, RAW264.7cells were transfected with miR-146a mimics or inhibitor, the mRNA and protein level of IRAK-1and TRAF-6were measured by quantitative RT-PCR and Western Blot. Compared with control group, macrophage transfected with miR-146a mimics showed a remarkably decrease of IRAK-1and TRAF-6in both mRNA and protein levels. To further address the possibility that the inhibition of IRAKI or TRAF6by miR-146a is responsible for the reduction of cytokines, we assessed the impact of IRAKI and TRAF6silencing by RNA interference. Subsequently, IRAK1/TRAF6silencing resulted in the drastically decrease of the above pro-inflammatory cytokines, TNF-α, IL-1β, IL-6and MCP-1, similar to the effect observed in RAW264.7cells with enforced expression of miR-146a. Meanwhile, si-IRAK-1and TRAF-6could promote BCG replication in macrophages. However, IRAK-1and TRAF-6had no effect on BCG internalization. Overall, these results suggested that overexpression of miR-146a can reduce the M.bovis BCG-induced pro-inflammatory cytokines by targeting IRAKI and TRAF6.In conclusion, miR-146a expression was strongly up-regulated upon M.bovis BCG infection and played a critical role in the infection. It can promote more bacillus invading into macrophages and simultaneously damage macrophage intracellular killing function so that tuberculosis could survival and proliferation in macrophage instead of been killed.