Cataloguing of Potential HIV Susceptibility Factors during the Menstrual Cycle of Pig-Tailed Macaques by Using a Systems Biology Approach

Abstract

Our earlier studies with pig-tailed macaques demonstrated various simian-human immunodeficiency virus (SHIV) susceptibilities during the menstrual cycle, likely caused by cyclic variations in immune responses in the female genital tract. There is concern that high-dose, long-lasting, injectable progestin-based contraception could mimic the high-progesterone luteal phase and predispose women to human immunodeficiency type 1 (HIV-1) acquisition and transmission. In this study, we adopted a systems biology approach employing proteomics (tandem mass spectrometry), transcriptomics (RNA microarray hybridization), and other specific protein assays (enzyme-linked immunosorbent assays and multiplex chemokine and cytokine measurements) to characterize the effects of hormonal changes on the expression of innate factors and secreted proteins in the macaque vagina. Several antiviral factors and pathways (including acute-phase response signaling and complement system) were overexpressed in the follicular phase. Conversely, during the luteal phase there were factors overexpressed (including moesins, syndecans, and integrins, among others) that could play direct or indirect roles in enhancing HIV-1 infection. Thus, our study showed that specific pathways and proteins or genes might work in tandem to regulate innate immunity, thus fostering further investigation and future design of approaches to help counter HIV-1 acquisition in the female genital tract.

Importance: HIV infection in women is poorly understood. High levels of the hormone progesterone may make women more vulnerable to infection. This could be the case during the menstrual cycle, when using hormone-based birth control, or during pregnancy. The biological basis for increased HIV vulnerability is not known. We used an animal model with high risk for infection during periods of high progesterone. Genital secretions and tissues during the menstrual cycle were studied. Our goal was to identify biological factors upregulated at high progesterone levels, and we indeed show an upregulation of genes and proteins which enhance the ability of HIV to infect when progesterone is high. In contrast, during low-progesterone periods, we found more HIV inhibitory factors. This study contributes to our understanding of mechanisms that may regulate HIV infection in females under hormonal influences. Such knowledge is needed for the development of novel prevention strategies.

FIG 1

Hierarchical clustering analysis of differentially expressed proteins from cervicovaginal lavage samples obtained from the luteal or follicular phase of the menstrual cycle. Proteins (n = 50) which were differentially abundant between menstrual phase samples according to the Student t test (P < 0.05) are illustrated. Protein details can be obtained at http://www.uniprot.org; hierarchical clustering of proteins was generated by unsupervised centroid linkage using the Pearson correlation as the distance metric. The abundance of each protein is shown in color (red color denotes overabundant proteins, yellow unchanged, and blue underabundant compared to the mean). Two trees (top) are observed distinguishing menstrual-phase samples (follicular grouping on left side, luteal on the right), with only 3 of the samples having heterogeneous protein abundance patterns (CV85 luteal; PPK1 and PHQ1 follicular). The follicular and luteal phases are distinguished by two distinct branches; the top branch (branch 2) groups overabundant proteins from the luteal phase, and the bottom branch (branch 1) shows those overabundant in the follicular phase. The macaque identifiers (example: CV85) are indicated at the top.

FIG 2

The major canonical pathways associated with differentially abundant proteins observed in the follicular and luteal phase. (A) The top two branches identified by hierarchical clustering were further analyzed using IPA pathway software. The major canonical pathways associated with branch 1 (magenta; top: follicular overabundant proteins) and branch 2 (orange; bottom: luteal overabundant proteins) are shown in decreasing order by significance value (P < 0.05). Numbers adjacent to charts (right side) indicate numbers of proteins overexpressed out of the total number of factors involved in this pathway as characterized by the IPA knowledgebase. A right-tailed Fisher's exact test (Benjamini-Hochberg corrected; horizontal axis represents −log10 P value) was used to assess the association between each protein appearing in the data set and a known canonical pathway. Only the top pathways (and those with a −log10 P value of 2.0 and at least 2 proteins/pathway) are shown for vertical sizing. (B) Spider plot illustrating the expression patterns of the top three associated pathways. The average expression profile of all proteins identified in the mass spectrometry data set belonging to these three pathways is shown. Each concentric circle represents the average log2-fold change of the specific protein for each menstrual phase (follicular phase, magenta; luteal phase, orange). The black dotted circle represents mean expression across both phases. Overexpression of these three pathways is shown in the follicular phase compared to the luteal phase.

FIG 3

Comparison of MCP-1 levels in the two menstrual phases of pig-tailed macaques, determined by Luminex technology; we used a random-effects model to evaluate differential expression (P < 0.05). A left-censoring mechanism was used in statistical analyses to handle observations below the limit of detection, shown here as zero.

FIG 4

(A) Volcano plot depicting distribution of fold change and statistical significance in the microarray data set. Data points in red indicate genes defined to be differentially expressed based on a 1.5-fold difference between phases and a P value of <0.05. (B) Heat map of 763 DEGs. The color scale is shown at the bottom; red indicates genes with higher expression in the luteal phase relative to the paired follicular-phase sample, and blue indicates higher relative expression in the follicular phase.

FIG 5

(A) Heat maps of genes showing luteal phase overexpression, organized into functional families as identified in the literature. The order of macaques is the same as that seen in Fig. 4B; red indicates genes with higher expression in the luteal phase relative to the paired follicular sample, and blue indicates higher relative expression in the follicular phase. (B) Gene set enrichment plots (GSEA) of two gene families enriched in the data set, TGF-β signaling and integrins, showing luteal-phase overexpression. The running enrichment score (y axis) is plotted by each gene's individual rank (x axis); bars below the x axis indicate individual gene ranks in the whole data set. The ranking is based on relative gene expression in the two menstrual phases. Genes in the leading edge (contributing the most to the enrichment score) are shown in red. The mean log2 probeset intensity of selected leading-edge genes is shown in the lower graphs. (C) Heat maps of genes showing follicular-phase overexpression, organized into functional families from the literature. (D) GSEA plot of interferon-stimulated gene set enriched in the data set, showing follicular phase overexpression. The mean log2 probeset intensity of selected leading-edge genes is shown on the right. The gene set chosen (Zhang interferon response) for GSEA was from the Broad Institute database.