Signature genes associated with immunological non-responsiveness to anti-retroviral therapy in HIV-1 subtype-c infection

Abstract

Objective: HIV-infected individuals undergoing therapy may show an immunological-discordant response to therapy, with poor CD4+ T cells recovery, despite viral suppression below the detection limit. The present study was carried out to delineate the underlying molecular mechanisms of immunological non-responsiveness to HIV therapy.

Design: We conducted microarray-based whole gene expression profiles of 30 subjects infected with HIV-1 subtype C, in peripheral blood to discern the signature genes associated with immunological non-responsiveness. After a thorough analysis and comparison of gene-expression profiles, microarray data was validated via qRT-PCR approach.

Results: Overall, we found 10 genes significantly up-regulated and 60 genes down-regulated (≥2-fold change) in immunological non-responders as compared to responders. Based on these results and pathway analysis of the protein-protein interaction, 20 genes were shortlisted for validation in human infected cases. We found statistically significant differences in expression levels of twelve genes IL-1α, IL-1β, IL-7R, TNF-α, FoxP3, PDCD5, COX7B, SOCS1, SOCS3, RPL9, RPL23, and LRRN3 respectively among immunological non-responders compared to therapy responders, confirming their an intimate relationship with immunological responsiveness to therapy.

Conclusions: Altogether, microarray and qRT-PCR validation results indicated that the aberrant expression of key genes involved in the regulation of T cell homeostasis, immune activation, inflammatory cytokine production, apoptosis, and immune-regulatory processes are possibly associated with immunological non-responsiveness in HIV-1 C infected individuals on ART.

Fig 1. Venn diagram showing the downregulated and upregulated genes.

Briefly, among HIV infected, overall, 16 probe sets (10 genes) significantly up-regulated and 71 probe sets (60 genes) down-regulated (>2-fold change) in ART-IF as compared to ART-R.

Fig 2. Heatmap and hierarchical clustering of genes in studied groups.

Hierarchical clustering (Hcl) expression image is shown. It is also clear from the above Hcl expression image i.e. ART-IF and ART-R clustered together. Red color shows over-expressed genes (>2.0) & Blue color shows under-expressed genes (<2.0). (The Hcl expression plot has been generated based on log2 normalized intensity value).

Fig 3. Volcano plots showing the distribution of significantly upregulated and downregulated genes.

Volcano plots were constructed by plotting the negative log of the p-value on the y-axis (-log10). The x-axis is the log of the log fold change among different groups. Distribution of downregulated and upregulated genes among ART-IF, ART-R, ART-N, and HCs.

Fig 4. Interactive network of the regulatory molecules.

String analysis was performed to study the protein-protein interaction. Downregulation of proteins differentially expressed in immunological failure HIV-1 subtype-C patients as compared to ART-R. Briefly, protein-protein interaction revealed four clusters with distinct biological functions. Group 1: Molecules involved in viral transcription, viral genome expression, and translation termination or inhibition. Group 2: Molecule involved in the cell cycle process. Group3: CD4-positive, alpha-beta T-cell differentiation, and activation. Lymphoid enhancer-binding factor 1 (LEF-1). Group 4: Integrin-mediated signaling pathway.

Fig 5. qRT-PCR analysis and validation of microarray gene expression data.

Differentially expressed genes among study groups (ART-IF, ART-R, and ART-N) were shortlisted for validation. Scatter plots showing the fold change in expression profile for IL-1α, IL-1β, IL-7R, TNF-α, FoxP3, PDCD5, COX7B, SOCS1, SOCS3, LRRN3, RPL9, and RPL23 genes by qRT-PCR (n = 10 each group). Significant values are indicated by star (*p<0.05, **p<0.01, ***p<0.001).