Transcriptome analyses identify key cellular factors associated with HIV-1-associated neuropathogenesis in infected men

Abstract

Objective: HIV-1 viral proteins and host inflammatory factors have a direct role in neuronal toxicity in vitro; however, the contribution of these factors in vivo in HIV-1-associated neurocognitive disorder (HAND) is not fully understood. We applied novel Systems Biology approaches to identify specific cellular and viral factors and their related pathways that are associated with different stages of HAND.

Design: A cross-sectional study of individuals enrolled in the Multicenter AIDS Cohort Study including HIV-1-seronegative (N = 36) and HIV-1-seropositive individuals without neurocognitive symptoms (N = 16) or with mild neurocognitive disorder (MND) (N = 8) or HIV-associated dementia (HAD) (N = 16).

Methods: A systematic evaluation of global transcriptome of peripheral blood mononuclear cells (PBMCs) obtained from HIV-1-seronegative individuals and from HIV-1-positive men without neurocognitive symptoms, or MND or HAD was performed.

Results: MND and HAD were associated with specific changes in mRNA transcripts and microRNAs in PBMCs. Comparison of upstream regulators and TimePath analyses identified specific cellular factors associated with MND and HAD, whereas HIV-1 viral proteins played a greater role in HAD. In addition, expression of specific microRNAs - miR-let-7a, miR-124, miR-15a and others - were found to correlate with mRNA gene expression and may have a potential protective role in asymptomatic HIV-1-seropositive individuals by regulating cellular signal transduction pathways downstream of chemokines and cytokines.

Conclusion: These results identify signature transcriptome changes in PBMCs associated with stages of HAND and shed light on the potential contribution of host cellular factors and viral proteins in HAND development.

Figure 1. Comparison of differentially regulated genes (q<0.05, FDR adjusted) in HIV-1 seropositive individuals without HAND, with MND or HAD relative to HIV-1 seronegative patients

(A) Schematic representation of relationship between different groups included in the study. HIV-1 seronegative individuals upon HIV-1 infection become HIV-1 seropositive and they can either be symptom free (no HAND) or can develop ANI, MND or HAD. The Venn diagram displays the number and overlap of significantly differentially expressed gene transcripts (q<0.05, FDR adjusted) among HIV-1 seropositive subjects without HAND symptoms, with MND or HAD compared to HIV-1 seronegative group. Total RNA isolated from PBMCs in these donors was characterized by Illumina HT-12 V4 array beadchips and the data were analyzed using genome studio. Multiple probes for a single gene were combined together. A detection cut-off of p <0.01 was used to identify transcripts that were detected consistently in each group. The expression of RNA transcripts were compared between HIV-1 seropositive individuals with MND and without HAND (B) or with HAD and without HAND (C). Pie chart denotes the number of RNA transcripts consistently detected in both the comparing groups (blue), or detected only in comparing HAND positive group (red) or detected only in those without HAND (green) or not detected in both the comparing groups (yellow). The volcano plot represents the fold change in expression of RNA transcripts in HAND positive group over no HAND group for only those genes that are consistently detected in both the comparing groups. Dotted vertical line (red) corresponds to a 2 fold cut-off and the dotted horizontal line (green) corresponds to p=0.05. (D) The Venn diagram displays the number and overlap of significantly differentially expressed gene transcripts (q<0.05, FDR adjusted) among the MND and HAD relative those without HAND. Red upward arrow indicates the increase and the green downward arrow indicates the decrease in number of the gene transcripts. Comparison of gene transcript expression unique for these HIV-1 seropositive individuals with or without HAND identified multiple upstream regulators including - (E) Cytokines and growth factors, (F) Factors involved in signal transduction – kinases, phosphatase, ligand associated nuclear receptors, membrane associated receptors, and (G) Transcription factors.

Figure 2. Network of miRNA-mRNA interaction and their associated functional role relevant in HAND pathogenesis

(A) Results from miRNA and mRNA array were combined by IPA based miRNA target analysis and visualized by Cytoscape. Experimentally verified miRNAs (inner circles) that actively regulate mRNA (outer group of circles) based on previously published literature are included. Green and red in the mRNA or miRNAs sets represents up and down regulation, respectively, in HIV-1 seropositive individuals with HAD relative to HIV-1 seropositive individuals without HAND symptoms. (B) Comparison of gene transcript expression unique for HIV-1 seropositive individuals with or without HAND relative to HIV-1 seronegative individuals identified miRNAs as key regulators in different stages of HAND. (C) Network denoting specific miRNAs and their targets identified differentially regulated in HAD relative to HIV-1 seropositive subjects without HAND (q<0.05, FDR adjusted) that are critical in chemokine signaling. (D) Network analysis of miRNA-mRNA pairs that are significantly dysregulated in HIV-1 seropositive subjects without HAND relative to HIV-1 seronegative subjects and their relation to downstream signaling molecules of multiple pro-inflammatory pathways. Pathways related to CSF2, CXCL8, TLR4, IL1β, RELA, TNF and p38 MAPK and only their associated miRNA-mRNA targets are shown.

Figure 3. Dynamic signaling and regulatory network for HIV-1 viral proteins in HAND associated changes in PBMC

Blue nodes are intermediate signaling proteins and green nodes are the TFs that are predicted to directly up/down-regulate the differential expression of target genes (targets not shown in figure, but the average levels of the regulated targets for each TF is presented by the yellow nodes while the size of each of the yellow nodes indicates how many genes belong to the cluster represented by the node). The figure displays the top predicted proteins for HAD stage and also demonstrated is the relation to the HIV-1 proteins via the signaling proteins and differentially expressed genes in earlier phases (The red nodes are the HIV-1 proteins [sources], see Fig S4, http://links.lww.com/QAD/B34, for additional details). Note that some intermediate proteins may also be TFs. The functional role in the network figure is based on the location of the protein in the selected paths based on the IP.

Figure 4. Transcriptome changes in PBMC related to HAND pathogenesis and their key regulators

Schematic representation of the relation of transcriptome changes in PBMC and in brain cells induced by neurotoxic, neuroprotective, viral factors and other factors derived from other compartments such as gastrointestinal (GIT) system, respiratory tract, bone marrow (BM) and others. Peripheral blood mononuclear cells in the brain microvasculature are exposure to diverse factors that play critical role in HAND pathogenesis. Evaluation of global transcriptome changes in all the populations of PBMCs, enriches transcriptome changes that occur across diverse cell types, including those transcriptome changes which are overlapping with diverse cells of the central nervous system compartment. Systematic analysis of these transcriptome changes in different stages of HAND identified key regulators associated with different stages of HAND. (−, denotes negative effect, +/−, denotes no effect, +, ++, and +++, denotes mild, moderate and severe positive effects, respectively). N-neuron, A-as strocyte, MG-microglia, MΦ-macrophage, SC- neuronal support cells, BMEC-brain microcapillary endothelial cells, BaM-basement membrane, Foot- foot of astrocyte, P-pericytes, BBB-blood brain barrier.