Mechanism Of Ipr1-mediated Macrophage Resistance To Mycobacterium Tuberculosis

Tuberculosis (TB) is a chronic and infectious disease caused by Mycobacterium tuber-culosis (MTB). As a zoonosis, TB strongly threatens human heath worldwide and causedseverely economic loss to cattle industries. About1/3of the human population were infectedwith MTB, however, only1/10infectants develop to active pulmonary tuberculosis, indicat-ing that the hereditary factors are essential for host resistance to TB. Located on thechromosome1of mouse, Ipr1have been proved to inhibit multiplication of intracellular pa-thogens such as MTB and Listeria monocytogenes, and to switch the apoptotic pathways ofinfected macrophages. The closest homologue of Ipr1in human is SP110, genetic polymor-phisms of SP110are associated with TB susceptibility in human and holstein-friesian cattle.These results suggested that Ipr1is a pivotal gene for host resistance to TB, and elucidationof the mechanisms of Ipr1-mediated resistance to MTB is significance for prevention andtreatment of tuberculosis, as well as disease-resistant animal breeding. However, to date, themechanism of Ipr1-mediated resistance to MTB is unclear. In this study, we investigate themechanism of Ipr1function by analyzing Ipr1protein interactome and the gene expressionprofile of Ipr1overexpressing macrophages. The main works and results were as follows,1. Analysis of the Ipr1protein interactome in mouse macrophage RAW264.7cells. Weconstructed RAW264.7cells stably expressing biotinylated Ipr1and designated asRAW-BAP-Ipr1-P2A-BirA, and control cells RAW-BAP-Ipr1and RAW-BAP-P2A-BirA.After verification of biotinylation of Ipr1, the function of biotinylated and unmodifiedIpr1were compared by immunofluorescence, coimmunoprecipitation (co-IP) and apoptoticanalysis. The results demonstrated that biotinylated Ipr1had similar function to the unmodi-fied Ipr1. Subsequently, we isolated Ipr1protein complexes by streptavidin-mediated affinitypurification and sequenced them using mass-spectrometer. A total of253Ipr1interactingproteins were identified by mass-spectrometric analysis, of which251are new candidates ofIpr1interacting proteins found in this study. The interaction of Ipr1and Vcp, Hspa5, Pcna,Rps3a, Ybx1, Vav1, Anxa2, Ncl, Prdx1, Wwox, Eif4g2, Atp2a2, Elavl1, Bag2, Oasl1, Mcm3was confirmed by co-IP. Functional annotation of identified Ipr1interacting proteins showed substantial enrich-ment in translation, ribosome biogenesis, RNA processing, mRNA metabolic process, proteinlocalization and DNA packaging. KEGG pathway analysis confirmed that Ipr1-interactingproteins were enriched in ribosome, spliceosome, DNA replication, Fc gamma R-mediatedphagocytosis and endocytosis pathways. These results indicated the unexplored functions ofIpr1except for apoptosis induction. Further investigation demonstrated that Ipr1-repressedglobal protein synthesis was not attributed to the enhanced phosphorylation of Eif2α. The in-teraction between Ipr1and endoplasmic reticulum (ER) stress marker Hspa5indicated thatIpr1may affect ER stress.2. Analysis of the regulation of Ipr1on gene transcription in RAW264.7cells by RNAsequencing (quantification)(RNA-seq(Q)). The RAW264.7cells stably expressing full lengthor truncated Ipr1gene were constructed by lentiviral transduction. Global transcriptomeanalysis of RAW-Ipr1and RAW-Control cells with or without the H37Ra infection was in-vestigated. The results showed that297genes had1.5-fold higher transcript abundance inRAW-Ipr1cells and483genes exhibited1.5-fold higher expression in RAW-Control cells(p<0.05). MTB infection changed a large number of gene expression in macrophages. A totalof1,735genes showed higher than1.5-fold changes in the RAW-Control cells infected withH37Ra compared with those in the uninfected RAW-Control cells, in which716genes wereupregulated and1019genes were downregulated by MTB. MTB infection caused more geneexpression changes in RAW-Ipr1cells than those in RAW-Control cells when compared withtheir uninfected counterparts, H37Ra infection upregulated808genes and downregulated1031genes in RAW-Ipr1cells.The functional annotation of differentially expressed genes according to GO terms re-vealed that Ipr1-regulated genes were highly enriched for terms associated with immuneresponse, defense response, cell proliferation regulation, response to wounding and apoptosisregulation. In contrast, MTB-regulated genes were mainly involved in cell cycle, cellular re-sponse to stress, death, apoptosis regulation and immune response.The comparison between Ipr1-regulated genes (DEG1) and MTB-regulated genes(DEG2) revealed that152genes exhibited opposite expression patterns, including cytokine,cytokine receptor and chemokine. These genes were significantly enriched for the GO termsrelated to immune response, wounding response, inflammatory response and defense re-sponse. These results indicated that Ipr1alters the immune response of host cells in responseto MTB infection by antagonizing MTB-inducing gene expression, particularly cytokine andchemokine, to protect host cells from MTB infection. The comparison between DEG2and DEG3found that755differentially expressedgenes were specifically in DEG3, which were not observed in DEG2. The differentially ex-pressed genes specifically in DEG3were mainly enriched in biological processes of immuneresponse, cell death, apoptosis, stress response, response to DNA damage and regulation oftranscription. These results indicated that Ipr1may regulate immune response and stress re-sponse when macrophages were infected with MTB, thus enhancing the macrophagesresistance to MTB.3. Analysis of miRNA expression profile in response to Ipr1and MTB infection bySmall RNA sequencing. We observed that21miRNAs were significantly upregulated and36miRNAs were downregulated (>1.5-fold, p<0.05) in RAW-Ipr1cells compared with those inRAW-Control cells. In contrast, H37Ra induced47miRNAs and inhibited14miRNAs inRAW264.7cells. The expression of miR-99b-let-7e-125a cluster and miR-21a were downre-gulated by Ipr1under both uninfected and H37Ra infected conditions. In this study, we foundthat miR-146a and miR-155increased in RAW-Ipr1cells compared with those inRAW-Control cells. Furthermore, MTB-infected RAW-Ipr1cells showed higher miR-146aand miR-155transcriptions than the infected RAW-Control cells. The expression of miR-27band miR-29a induced by H37Ra were significantly downregulated in H37Ra-infectedRAW-Ipr1cells. These results demonstrated the possibility that Ipr1enhances apoptosis andMTB killing ability of host cells via regulating miRNA expression, thus alters the amount ofmiRNA’s targets.4. Construction of the model of Ipr1-mediated macrophage resistance to MTB. In thisstudy, we predicted the targets of miR-125a, miR-99a, miR-99b, miR-342and let-7e via in-tegrating of our sequencing data and bioinformatics analysis. Furthermore, we validated thepredicted miRNA-mRNA interaction by luciferase reporter assays. The results demonstratedthat miR-125a inhibited Bmf expression via interacting with Bmf3’UTR. In addition, ourdata also demonstrated that overexpression of Bmf induced apoptosis of RAW264.7cells.In this study, we examined the UPR indicators in RAW-Ipr1and RAW-Control cellswith or without MTB infection to validate whether Ipr1could affect the ER stress response.Remarkably, Hspa5and Hspa8were significantly increased in RAW-Ipr1cells, indicating theUPR signaling was activated by Ipr1. However, CHOP and phosphorylation of Eif2α weresignificantly suppressed in RAW-Ipr1cells. This phenomenon is contradictory but consistentwith the adaptation to ER stress. We speculated that the macrophages stably expressing Ipr1could adapt to ER stress because the UPR pathways lead to death of highly activated cellsduring the screening process. Further studies demonstrated that transient expression of Ipr1indues ER stress, and the likely mechanism is alterlation of the subcellular localization of Hspa5by Ipr1via protein-protein interaction. In addition, our study suggested that inductionof ER stress is essential for Ipr1-mediated apoptosis of macrophages after MTB infection.Based on our results, we speculated that Ipr1-mediated macrophage resistance to TBthrough multiple manners. On the one hand, Ipr1competitively binds to Hspa5and sequest-ers Hspa5in the nucleus, induceing ER stress and initiating the UPR signaling cascades andupregulating the downstream target of UPR, such as CHOP and chaperones. CHOP can pro-mote apoptosis. On the other hand, Ipr1regulated mRNA and miRNAs, such as Bmf, Pdcd1,Pdcd4, Tnf, Il10, miR-146a, miR-155, miR-125a, miR-21a and miR-99b, and thus initiates orenhances the apoptotic cell death and mycobacterial killing.