Transcriptome analysis of long non-coding RNAs in Mycobacterium avium complex-infected macrophages

Mycobacterium avium complex (MAC) is a non-tuberculosis mycobacteria widely distributed throughout environment. Even though the MAC infection is currently increasing in old women and patients with affected immune system, comprehensive analysis of MAC-infected host cell transcriptome, in particular that of long non-coding RNAs (lncRNAs), has scarcely been reported. Using in vitro cultured primary mouse bone marrow-derived macrophages (BMDMs) and CAGE technology, we analyzed the transcriptional and kinetic landscape of macrophage genes, with focusing on lncRNAs, during MAC infection. MAC-infected macrophages induced immune/inflammatory response genes and other genes as similar as M1 activation, consistent with previous reports, although Nos2 and Arg1 revealed distinct expression profiles from M1 activation. We identified 31 up-regulated and 30 down-regulated lncRNA promoters corresponding to 18 and 27 lncRNAs, respectively. The up-regulated lncRNAs were clustered into two groups, early and late up-regulated groups, which were predicted to associate with the immune activation and the immune response to infection, respectively. Further, the Ingenuity IPA analysis predicted canonical pathways and upstream transcription regulators associated with differentially expressed lncRNAs. Several differentially expressed lncRNAs that have been reported elsewhere revealed various expressional change by M1 or M2 pre-activation and the following MAC infection. Finally, we showed that expressional change of lncRNAs in MAC-infected BMDMs is predominantly mediated by Tlr2, but there may be other weakly sensing mechanism for MAC infection. Taken together, we identified differentially expressed lncRNAs in MAC-infected BMDMs, which revealed diverse features implying their distinct role in MAC infection and macrophage polarization. Overall design: Bone marrow cells, generated from 8-12 week old BALB/c male mice, were harvested from the femurs. Cells were cultured for 10 days at 37 oC under 5% CO2 in RPMI-1640 containing 10% FBS, 40 ng/ml GM-CSF, 20 ng/ml M-CSF, and 50 µ/ml ampicillin in 90 mm x 15 mm petri dishes with vent. After 10 days, bone marrow-derived macrophages (BMDMs) were harvested and were plated in 6-well plates at 2 x 106 cells per well overnight. BMDMs were infected with log phase Mycobacterium avium subsp. hominissuis (MAH) strain TH-135 (MOI = 200) for 4 hours. Cells were washed to remove extracellular mycobacteria, replenished with fresh medium, and incubated for 0, 4, 12, and 24 hours post infection. For M1 or M2 pre-activation experiments, BMDMs were plated in 6-well plates at 2 x 106 cells per well overnight, followed by stimulation with IFN? (100 unit/ml) or IL-4/IL-13 (100 units/ml each) for 24 hours. Cells were infected with MAH for 4 hours, washed to remove extracellular mycobacteria, and incubated for 24 hours post-infection. Quadruplicate samples were produced at each condition.