Function Analysis Of SIRPα And LincRNA-Cox2 In Mtb-induced Inflammatory Response

Tuberculosis is now about to come of age as a global emergency. Much progress has been made in the control of tuberculosis, but it remains a serious and substantial threat to the health of people worldwide, causing 1.3million unnecessary deaths every year, more than 30 million people have died from tuberculosis in the past 21 years.The complex molecular events that occur within the host during the establishment of Mycobacterium tuberculosis infection are poorly defined,thus preventing identification of predictive markers of disease progression and state. To identify such molecular markers during M.tuberculosis infection, global changes in transcriptional response in the host were assessed using mouse whole genome arrays. The main resulrs as follows:1、Screening and identification of differential expressed genes and proteins in Mtb-infected macrophages.The RAW264.7 cells model infected by BCG were established.The expression profile of cellular genes during BCG infection was analyzed by microarray.Then SIRPα and LincRNA-Cox2 which expression were significantly altered were screened and identified in several infection models as in BCG infected RAW264.7 cells and TB patients by the quantitative RT-PCR \Western blot and Immunohistochemistry.2、To examine SIRPα’s regulation of the innate immune responses during BCG infection, we first used SIRPα-KD(knockdown)and VT(vector) macrophages respectively. Colony forming units(CFU) count shows that, compared with SIRP-VT cells, SIRPα-KD significantly inhibit BCG’s survival in macrophages.3 、 Here we study the stimulation of BCG in macrophages causes mycobacterial phagosomes to mature into phagolysosomes. In response to BCG, knocking down SIRPα also resulted in mycobacterial phagosome colocalization with the autophagy effector LC3, an elongation factor in autophagosome formation. After stimulation with BCG, SIRPα-KD macrophages significantly increased phagosomal colocalization with Beclin-1, a subunit of the phosphatidylinositol 3-kinase h VPS34, necessary for autophagy and a target for mycobacterial phagosome maturation arrest.Induction of autophagy suppressed intracellular survival of mycobacteria.4、In response to BCG infection, SIRPα-KD macrophages were found to exhibit enhanced phosphorylation of p38 MAPK in RAW cells rather than JNK and ERK passways. The finding demonstrate that SIRPαnegatively regulates BCG signaling by suppressing the MAPKs pathways in macrophage. We then study the effects of SIRPαon the production of inflammatory cytokines. SIRPα-KD macrophages produced significantly more TNF-α、IL-6 and IL-12 than VT macrophages.SIRPαknockdown also significantly raised BCG-induced Cox-2 production and mRNA expression in RAW cells.5 、 In the research of LincRNA-Cox2,real-time PCR showed that the expression of LincRNA-Cox2 was significantly increased after infected with BCG in RAW264.7 cells. After inhibiting p38 MAPK and JNK signal pathways in RAW264.7 cells infected with BCG, the expressions of Linc RNA-Cox2 were significantly decreased.After down-regulating Linc RNA-Cox2, the expression of TNF-α was significantly reduced.In one world, our data clearly indicate that The knockdown of SIRPαinduced autophagic pathways can overcome the trafficking block imposed by M. Tuberculosis. Taken together, SIRPαregulates the expression profile of proinflammatory cytokines through both autophagy and MyD88-dependent pathway.LincRNA-Cox2 may induce the increasing ofTNF-α to suppress intracellular survival of BCG in a p38 MAPK and JNK-dependent manner.