Peripheral blood mononuclear cell transcriptomes reveal an over-representation of down-regulated genes associated with immunity in HIV-exposed uninfected infants

Abstract

HIV-exposed uninfected (HEU) infants are disproportionately at a higher risk of morbidity and mortality, as compared to HIV-unexposed uninfected (HUU) infants. Here, we used transcriptional profiling of peripheral blood mononuclear cells to determine immunological signatures of in utero HIV exposure. We identified 262 differentially expressed genes (DEGs) in HEU compared to HUU infants. Weighted gene co-expression network analysis (WGCNA) identified six modules that had significant associations with clinical traits. Functional enrichment analysis on both DEGs and the six significantly associated modules revealed an enrichment of G-protein coupled receptors and the immune system, specifically affecting neutrophil function and antibacterial responses. Additionally, malaria pathogenicity genes (thrombospondin 1-(THBS 1), interleukin 6 (IL6), and arginine decarboxylase 2 (ADC2)) were down-regulated. Of interest, the down-regulated immunity genes were positively correlated to the expression of epigenetic factors of the histone family and high-mobility group protein B2 (HMGB2), suggesting their role in the dysregulation of the HEU transcriptional landscape. Overall, we show that genes primarily associated with neutrophil mediated immunity were repressed in the HEU infants. Our results suggest that this could be a contributing factor to the increased susceptibility to bacterial infections associated with higher morbidity and mortality commonly reported in HEU infants.

Figure 1

Hierarchical clustering of the top 50 differentially expressed genes (up- and down-regulated). Red; relative increase in gene expression, blue; relative decrease in gene expression. x-axis; sample identification, y-axis; genes identified.

Figure 2

Volcano plot demonstrating the differentially expressed genes in HEU and HUU infants. The x-axis represents the log₂ fold change while the y-axis represents the −log₁₀ of BH adjusted p-value (padj) value. The differentially expressed genes are indicated in red (padj, 0.05 & L₂FC > 2).

Figure 3

Functional enrichment analysis of the differentially expressed genes (DEGs). The y-axis represents enriched GO terms (FDR < 0.01) associated with DEGs in HEU infants sorted by biological processes, cellular component “Cellular C.” and molecular function “Molecular F.” The x-axis represents the absolute log₁₀ FDR values.

Figure 4

Antimalarial antibody titers in HEU and HUU infants. Antibody responses against (a) Plasmodium schizont extracts and (b) recombinant plasmodium apical membrane antigen 1 (PfAMA1). The Mann Whitney non-parametric test was used to compare antibody levels between HEU and HUU infants in high malaria transmission (HEU_high against HUU_high) and between HEU and HUU infants in low malaria transmission areas (HEU_low against HUU_low). Median of log transformed arbitrary antibody tires are shown and p < 0.05 considered significant. HEU; HIV exposed uninfected infants, HUU; HIV unexposed uninfected infants.

Figure 5

(A) Cluster dendogram of co-expression modules. The weighted gene co-expression network analysis identified 12 modules from hierarchical clustering of HEU/HUU co-expression profiles. (B) Hierarchical clustering of the module- trait relationship. The y-axis; modules and the x-axis; clinical traits. Pearson’s correlation co-efficient and p-values are shown. Red indicates positive regression while blue indicates negative regression. (C) Eigengene network dendogram representing the relationship between the modules and the two traits of interest; HEU vs HUU and neutrophils. (D) Gene ontology network representation of the five significant modules; Green, Black, brown, purple and green/yellow. (E) Expression analysis of genes involved in neutrophil mediated immunity. The x axis represents genes with significant involvement in neutrophil mediated immunity while the y-axis represents the normalized counts in HEU and HUU infants. *Asterics indicate genes with statistically significant differences (Mann Whitney non-parametric test). HEU; HIV exposed uninfected infants, HUU; HIV unexposed uninfected infants.

Figure 6

Pearson correlation analysis comparing the transcription profile between chromatin genes and DEGs. Red and blue color-coding indicates relative increase or decrease in gene expression profile, respectively. Differentially expressed genes and Chromatin genes are represented by the x and y axis respectively.