A study of the key genes and inflammatory signaling pathways involved in HLA-B27-associated acute anterior uveitis families

Abstract

The present study was conducted to investigate the key genes and the inflammatory signaling pathways involved in HLA-B27-associated acute anterior uveitis (AAU) families. Four families with HLA-B27‑positive aau patients and their HLA-B27-positive blood relatives were included in the study. peripheral blood monocytes were isolated from the subjects and stimulated by lipopolysaccharides (LPS). Gene expression microarrays were used to identify the differentially expressed genes (DEGs), and the DEGs were analyzed by a range of bioinformatics-based techniques, including Gene Ontology (GO), Pathway analysis, Signal-Net analysis and Gene Relation Network (Gene-Rel-Net). Finally, ELISA was used to quantify cytokines in the supernatant. The gene expression microarrays identified 801 DEGs, including 349 upregulated and 452 downregulated genes. The GO analysis revealed several important functions, including metabolic, immune and inflammatory responses. The pathway analysis highlighted the enhanced activity of Staphylococcus aureus infection, chemokine and metabolic signaling pathways, as well as cytokine-to‑cytokine receptor interactions. A total of 18 DEGs that were found to play critical roles by Signal-Net and Gene-Rel-Net and verified by quantitative polymerase chain reaction analysis were identified as key genes. In conclusion, monocytes from the AUU patients were more sensitive and exhibited a more prominent inflammatory response to stimulation by LPS compared with monocytes from healthy HLA-B27-positive blood relatives. These characterized DEGs may provide new evidence for the pathogenesis of AAU and help identify new therapeutic targets.

Figure 1

CD14-positive monocytes. Immunofluorescence staining of CD14 (green) was identified on the cell membrane. The nuclei were stained with 4′,6-diamidino-2-phenylindole (blue).

Figure 2

Significantly changed Gene Ontologies (GOs) of differentially expressed genes. (A) Up-GOs. (B) Down-GOs. The x-axis shows the negative logarithm of the P-value (-LgP) and the y-axis shows the GO category. A higher −LgP indicated a smaller P-value. A high −LgP-value was correlated with a greater significance of the GO category.

Figure 2

Significantly changed Gene Ontologies (GOs) of differentially expressed genes. (A) Up-GOs. (B) Down-GOs. The x-axis shows the negative logarithm of the P-value (-LgP) and the y-axis shows the GO category. A higher −LgP indicated a smaller P-value. A high −LgP-value was correlated with a greater significance of the GO category.

Figure 3

Significant changed pathways based on the KEGG database with P/0.05 and FDR/0.05. (A) Significantly upregulated pathways. (B) Significantly downregulated pathways. The y-axis shows the pathway category and the x-axis shows the negative logarithm of the P-value (-LgP). A higher -LgP indicated a smaller P-value. KEGG, Kyoto Encyclopedia of Genes and Genomes; FDR, false discovery rate.

Figure 3

Significant changed pathways based on the KEGG database with P/0.05 and FDR/0.05. (A) Significantly upregulated pathways. (B) Significantly downregulated pathways. The y-axis shows the pathway category and the x-axis shows the negative logarithm of the P-value (-LgP). A higher -LgP indicated a smaller P-value. KEGG, Kyoto Encyclopedia of Genes and Genomes; FDR, false discovery rate.

Figure 4

Signal-Net of differentially expressed genes. The red circles represent the upregulated genes and the blue circles represent the downregulated genes. The area covered by the circle represents the degree. Interaction between the genes is shown as: a, activation; a(p), activation (phosphorylation); c, compound; ex(ind), expression (indirect effect).

Figure 5

Gene relation network (Gene-Rel-Net) of differentially expressed genes. (A) Differentially expressed genes of HLA-B27-associated acute anterior uveitis. (B) Differentially expressed genes of HLA-B27-associated controls. The red circles represent the upregulated genes and the blue circles represent the downregulated genes. The area covered by the circle represents the degree.

Figure 5

Gene relation network (Gene-Rel-Net) of differentially expressed genes. (A) Differentially expressed genes of HLA-B27-associated acute anterior uveitis. (B) Differentially expressed genes of HLA-B27-associated controls. The red circles represent the upregulated genes and the blue circles represent the downregulated genes. The area covered by the circle represents the degree.

Figure 6

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) validation of 18 differentially expressed genes. (A) Difference between A1 and B1. (B) Difference between A2 and B2. Total RNA was extracted for RT-qPCR to analyze the expression of 18 important genes identified by Signal-Net analysis and Gene-Rel-Net. The results are shown as mean ± standard deviation of three independent experiments (P<0.05). The y-axis shows the relative mRNA expression and the x-axis shows the 18 important genes.

Figure 6

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) validation of 18 differentially expressed genes. (A) Difference between A1 and B1. (B) Difference between A2 and B2. Total RNA was extracted for RT-qPCR to analyze the expression of 18 important genes identified by Signal-Net analysis and Gene-Rel-Net. The results are shown as mean ± standard deviation of three independent experiments (P<0.05). The y-axis shows the relative mRNA expression and the x-axis shows the 18 important genes.

Figure 7

ELISA analysis of culture supernatants. The increased concentration of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 in the culture supernatants of monocytes isolated from the patients and their blood relatives folowing stimulation by lipopolysaccharides. Cytokine levels are expressed as mean ± standard deviation (vertical bars) in pg/ml.