Peripheral blood gene expression profiling in Sjögren's syndrome

Abstract

Sjögren's syndrome (SS) is a common chronic autoimmune disease characterized by lymphocytic infiltration of exocrine glands. The affected cases commonly present with oral and ocular dryness, which is thought to be the result of inflammatory cell-mediated gland dysfunction. To identify important molecular pathways involved in SS, we used high-density microarrays to define global gene expression profiles in the peripheral blood. We first analyzed 21 SS cases and 23 controls, and identified a prominent pattern of overexpressed genes that are inducible by interferons (IFNs). These results were confirmed by evaluation of a second independent data set of 17 SS cases and 22 controls. Additional inflammatory and immune-related pathways with altered expression patterns in SS cases included B- and T-cell receptor, insulin-like growth factor-1, granulocyte macrophage-colony stimulating factor, peroxisome proliferator-activated receptor-alpha/retinoid X receptor-alpha and PI3/AKT signaling. Exploration of these data for relationships to clinical features of disease showed that expression levels for most interferon-inducible genes were positively correlated with titers of anti-Ro/SSA (P<0.001) and anti-La/SSB (P<0.001) autoantibodies. Diagnostic and therapeutic approaches targeting interferon-signaling pathway may prove most effective in the subset of SS cases that produce anti-Ro/SSA and anti-La/SSB autoantibodies. Our results strongly support innate and adaptive immune processes in the pathogenesis of SS, and provide numerous candidate disease markers for further study.

Figure 1. Gene Expression Profiles in Two Independent Sjögren’s Syndrome Cohorts

Each row represents a single transcript and each column represents a single subject. SS cases (blue) and controls (orange) are indicated along the top of each cluster diagram and above each column of expression data. Horizontal bars on the right of each diagram indicate IFN-inducible genes (purple), previously defined by direct in vitro stimulation experiments or other data from the literature. Log₂ transformed ratios of individual expression values divided by the mean of the controls were calculated for each transcript. These values were used in hierarchical clustering analyses. Relative intensities are indicated for overexpressed (red) and underexpressed (green) transcripts. (A) Differentially expressed transcripts (n=425) for Cohort 1. (B) Differentially expressed transcripts (n=120) for Cohort 2.

Figure 2. Summary of statistically significant canonical pathways identified through IPA

Canonical pathways are listed across the top from left to right in order of statistical significance in Cohort 1 with P value ranges indicated. Pathways indicated in bold italics represent those showing significance in both Cohorts 1 and 2. The left most column lists differentially expressed genes initially grouped by structural category to show cellular localization (extracellular, plasma membrane, cytoplasm, or nucleus). The genes within each of the 4 structural categories are further organized by ranking each gene according to initial occurrence in the most significant canonical pathway as statistically ranked across the top from left to right. The color-coded boxes indicate the fold-change differences in mean expression levels for SS cases in Cohort 1 relative to controls.

Figure 3. Correlation of clinical features with gene expression profiles in SS

(A) Hierarchical clustering analysis of 223 differentially expressed transcripts between 36 SS cases (blue) and 22 controls (orange) in Cohort 3. Color-coding is as described in Figure 1. (B) Bar graphs showing the distribution of measurements for anti-Ro/SSA (blue) and anti-La/SSB (gold) autoantibodies as measured by ELISAs, tear flow measurements as measured by Schirmer’s Tests (ST; maroon) and whole unstimulated salivary flow (WUSF; green) for each individual in panel A. (C) Correlations between RNA transcript levels (rows) in panel A and clinical measurements of autoantibodies, tear flow, and salivary flow. Dashed lines indicate statistical significance thresholds (P=0.05) determined through permutation testing.