Regulation of global gene expression in human Loa loa infection is a function of chronicity

Abstract

Background: Human filarial infection is characterized by downregulated parasite-antigen specific T cell responses but distinct differences exist between patients with longstanding infection (endemics) and those who acquired infection through temporary residency or visits to filarial-endemic regions (expatriates).

Methods and findings: To characterize mechanisms underlying differences in T cells, analysis of global gene expression using human spotted microarrays was conducted on CD4(+) and CD8(+) T cells from microfilaremic Loa loa-infected endemic and expatriate patients. Assessment of unstimulated cells showed overexpression of genes linked to inflammation and caspase-associated cell death, particularly in endemics, and enrichment of the Th1/Th2 canonical pathway in endemic CD4(+) cells. However, pathways within CD8(+) unstimulated cells were most significantly enriched in both patient groups. Antigen (Ag)-driven gene expression was assessed to microfilarial Ag (MfAg) and to the nonparasite Ag streptolysin O (SLO). For MfAg-driven cells, the number of genes differing significantly from unstimulated cells was greater in endemics compared to expatriates (p<0.0001). Functional analysis showed a differential increase in genes associated with NFkB (both groups) and caspase activation (endemics). While the expatriate response to MfAg was primarily a CD4(+) pro-inflammatory one, the endemic response included CD4(+) and CD8(+) cells and was linked to insulin signaling, histone complexes, and ubiquitination. Unlike the enrichment of canonical pathways in CD8(+) unstimulated cells, both groups showed pathway enrichment in CD4(+) cells to MfAg. Contrasting with the divergent responses to MfAg seen between endemics and expatriates, the CD4(+) response to SLO was similar; however, CD8(+) cells differed strongly in the nature and numbers (156 [endemics] vs 36 [expatriates]) of genes with differential expression.

Conclusions: These data suggest several important pathways are responsible for the different outcomes seen among filarial-infected patients with varying levels of chronicity and imply an important role for CD8(+) cells in some of the global changes seen with lifelong exposure.

Figure 1. Volcano plots of differentially expressed genes.

Volcano plots of differentially expressed genes (p<0.01) in the unstimulated CD4⁺ (left panel) and CD8⁺ (right panel) T cells of filarial-infected expatriate (blue dots) and endemic (red dots) patients. Genes not significantly different are symbolized by black dots with each dot denoting a single gene. The x-axis is the fold difference (log 2) between patient groups and the y-axis represents the log10 of the p-value.

Figure 2. Quantitative RT-PCR and microarray correlation.

Correlation between Taqman™ qRT-PCR values and microarray expression values for 10 genes in CD4⁺ cells (top panel) and 7 genes in CD8⁺ cells (bottom panel). Each point illustrates the average value of the 3 individuals within each patient group for qRT-PCR (x-axis) vs microarray (y-axis) for the same sample. Quantitative RT-PCR values were calculated as the antilog of ([1/ΔCT]×100) where ΔCT = cycle threshold (CT) of the test gene minus the CT of the 18S ribosomal gene; microarray values are the antilog of the intensity values imported from GenePix. The Spearman Rank Correlation was used to calculate r- and p-values. Data associated with these plots are shown in Table S1.

Figure 3. Functional analysis of genes in unstimulated cells.

Number of genes associated with specific cellular functions in the unstimulated CD4⁺ (left panel) and CD8⁺ (right panel) T cells of Loa loa infected endemic (red bars) and expatriate (blue bars) patients. Annotation for cell functions was extracted from www.ingenuity.com using genes that were differentially expressed at p<0.01 and adjusted for multiple comparisons using the Benjamini Hochberg False Discovery Rate (FDR) correction.

Figure 4. Cell death network in unstimulated cells.

Network of differentially expressed molecules associated with cell death (www.ingenuity.com) in the unstimulated CD4⁺ and CD8⁺ T cells of endemic (in red) and expatriate (in blue) patients. Genes were differentially expressed at p<0.01, adjusted for multiple comparisons using the Benjamini Hochberg False Discovery Rate (FDR) correction. Lines represent direct (solid lines) and indirect (dashed lines) relationships between molecules and molecules within double circles represent complexes of genes.

Figure 5. Venn diagrams illustrating the number of Ag-driven genes.

Venn diagrams representing the number of genes either upregulated (upward facing arrows) or downregulated (downward facing arrows) to parasite (microfilarial Ag; MfAg, top panels) and nonparasite (streptolysin O; SLO, bottom panels) antigens in endemic (pink circles) and expatriate (blue circles) filarial-infected patients. CD4⁺ T cells are shown in the left panels and CD8⁺ T cells are in the right panels. Numbers represent genes that were significantly up-or down-regulated (p<0.001) in comparison to unstimulated cells.

Figure 6. Networks of microfilarial Ag-driven upregulated genes.

Two major networks (Figures A and B; www.ingenuity.com) of differentially expressed molecules from either CD4⁺ or CD8⁺ T cells that were significantly upregulated (p<0.001) to MfAg in endemic (in red) and expatriate (in blue) patients. Lines represent direct (solid lines) and indirect (dashed lines) relationships between molecules, and molecules within double circles represent complexes of genes.

Figure 7. Network of microfilarial Ag-driven downregulated genes.

Major network (www.ingenuity.com) of differentially expressed molecules that were significantly downregulated (p<0.001) to MfAg in either CD4⁺ or CD8⁺ T cells of endemic (in red) and expatriate (in blue) patients. Lines represent direct (solid lines) and indirect (dashed lines) relationships between molecules and molecules within double circles represent complexes of genes.