Study On The Transcriptome Of Tuberculosis-associated Immune Reconstitution Inflammatory Syndrome In HIV/AIDS Patients During Antiretroviral Therapy

ObjectiveThis study aims to determine all factors or markers related to Tuberculosis-associated Immune Reconstitution Inflammatory Syndrome(TB-IRIS) through the RNA-sequencing technology, and to explore early warning indicators for early diagnosis, and so as to reduce the incidence and mortality of TB-IRIS and to improve the life quality of patients with AIDS-TB co-infectious.MethodsFour cases of HIV(human immunodeficiency virus) infection only(group H), 6 cases of HIV co-infection with TB patients who did not have IRIS(group HT), and 6 cases of HIV co-infection with TB patients who did have IRIS(group HTI) were selected. Peripheral Blood Mononuclear Cells(PBMCs) were isolated at two different time points: before and two-weeks after the initiation of Antiretroviral Therapy(ART) treatment. c DNA libraries were constructed for the RNA-seq technology to examine the PBMC transcriptomes at this two samples points. The differentially expressed genes(DEGs) were identified by pairwise comparisons between 1) HT group(Before ART versus after), 2) HTI group(Before ART versus after), 3) HT group versus HTI group(Both before ART). Bioinformatics of enrichment analysis(Target Mine) was used to determine relevant pathways. Using the real-time fluorescent quantitative polymerase chain reaction(q-PCR), the sequencing results were validated. Identifications of marker genes of TB-IRIS were performed.Results(1) Before the initiation of ART, the CD4+ T-cell count was lower(p-value=0.010), coinciding with greater HIV-RNA load(p-value=0.023) in the HTI group than in the H and HT groups. After ART, in the HTI group, the CD4+ T-cell counts recovered to a certain degree and the HIV-RNA load significantly decreased(reduced to 1% of the original on average). However, in comparison to other two groups, the HIV-RNA load in the HTI group was still significantly higher.(2) For the comparisons of HT versus HTI before ART, 392 DEGs(197 up-regulated, 195 down-regulated)(p-value< 0.01, FDR< 0.05) were identified. Target Mine Pathway analysis show that 38 of 392 DEGs were enriched in the Generic Transcription Pathway(p-value=1.11E-6).(3) For the comparison between the HT groups(before and after ART), 36 DEGs were identified(28 up-regulated, 8 down-regulated), whereas for the comparison of the HTI groups(before and after ART), 74 DEGs were identified(50 up-regulated, 24 down-regulated). And for the comparison between the H groups(before and after ART), no DEG was detected. In the pathway analysis, 74 of the DEGs from the HTI group were significantly enriched in the Interferon alpha/beta signaling pathway(p-value=1.35E-7) and Cytokine-related pathways, such as Cytokine Signaling in Immune system(p-value=1.1E-3) and Cytokine-cytokine receptor interaction(p-value=1.9E-2).(4) We also found 88 DEGs(7 down-regulated, 81 up-regulated) after ART between the HT and HTI groups. Pathway analysis enriched the DEGs in Cell cycle, Mitotic, and Cell cycle checkpoints related pathways.Conclusions(1) Before ART, low CD4+ T cell counts and high HIV-RNA load were probably associated with the occurrence of TB-IRIS.(2) The Interferon alpha/beta signaling pathway, Cytokine-related pathways and Cell cycle pathways were related to the occurrence of TB-IRIS.(3) Successful establishment of gene expression profiles of TB-IRIS demonstrated that RNA-seq technology can be used to predict TB-IRIS.