Susceptibility to acute rheumatic fever based on differential expression of genes involved in cytotoxicity, chemotaxis, and apoptosis

Abstract

It is unknown why only some individuals are susceptible to acute rheumatic fever (ARF). We investigated whether there are differences in the immune response, detectable by gene expression, between individuals who are susceptible to ARF and those who are not. Peripheral blood mononuclear cells (PBMCs) from 15 ARF-susceptible and 10 nonsusceptible (control) adults were stimulated with rheumatogenic (Rh+) group A streptococci (GAS) or nonrheumatogenic (Rh-) GAS. RNA from stimulated PBMCs from each subject was cohybridized with RNA from unstimulated PBMCs on oligonucleotide arrays to compare gene expression. Thirty-four genes were significantly differentially expressed between ARF-susceptible and control groups after stimulation with Rh+ GAS. A total of 982 genes were differentially expressed between Rh+ GAS- and Rh- GAS-stimulated samples from ARF-susceptible individuals. Thirteen genes were differentially expressed in the same direction (predominantly decreased) between the two study groups and between the two stimulation conditions, giving a strong indication of their involvement. Seven of these were immune response genes involved in cytotoxicity, chemotaxis, and apoptosis. There was variability in the degree of expression change between individuals. The high proportion of differentially expressed apoptotic and immune response genes supports the current model of autoimmune and cytokine dysregulation in ARF. This study also raises the possibility that a "failed" immune response, involving decreased expression of cytotoxic and apoptotic genes, contributes to the immunopathogenesis of ARF.

FIG 1

Experimental design. PBMCs from 15 subjects in the ARF group and from 10 subjects in the control group were stimulated with Rh+ GAS and Rh− GAS for 0, 3, and 24 h, and samples were amplified. Amplified samples from 3 and 24 h were hybridized with samples from 0 h to two-color microarray slides. Comparisons were made between the subject groups (analyses 1 and 2) and between the types of bacteria (analyses 3 and 4) (see Materials and Methods).

FIG 2

Volcano plots of differences in gene expression following in vitro stimulation with Rh+ GAS between the ARF and control groups for 3 h (analysis 1) and 24 h (analysis 2). Each point represents a gene. Genes with positive values are those that were more highly expressed in the ARF group, and genes with negative values are those more highly expressed in the control group. Log fold change, log₂ change (M value); log odds, statistical significance (higher indicates more significant).

FIG 3

Interactions between genes that were differentially expressed after 24 h using a threshold difference in expression of 50%. Genes that were increased in the ARF group compared to the control group are shown in red, and genes that were decreased are shown in green, with the intensity of color reflecting the magnitude of differential expression.

FIG 4

Volcano plots of differential gene expression in the ARF group following in vitro stimulation with Rh+ GAS and Rh− GAS for 3 h (analysis 3) and 24 h (analysis 4). Genes with positive values are those that were more highly expressed with Rh+ GAS stimulation, and genes with negative values are those more highly expressed with Rh− GAS stimulation. Log fold change, log₂ change (M value); log odds, statistical significance.

FIG 5

Immune genes with significantly decreased expression after 24 h between in vitro stimulation with Rh+ GAS in ARF subjects and both Rh+ GAS in control subjects and Rh− GAS in ARF subjects. Each point represents the log₂ fold change in gene expression (M value) for one individual after stimulation; the horizontal bar represents the mean M value of the group. IFN-γ, gamma interferon; GZMB, granzyme B; GNLY, granulysin; IL-10, interleukin 10; PRF1, perforin 1; SLAMF1, signaling lymphocytic activation molecule family member 1. The one significantly increased gene (TYROBP) is not shown.