Immunosuppressant exposure confounds gene expression analysis in systemic lupus erythematosus

Abstract

Objectives: The analysis of gene module expression in SLE is emerging as a tool to identify active biological pathways, with the aim of developing targeted therapies for subsets of patients. Detailed information on the effect of immunosuppressants on gene module expression is lacking. We aimed to examine the impact of medication exposure on gene module expression.

Methods: A set of commercially available disease-relevant gene modules were measured in 730 whole blood samples from a dedicated lupus clinic on whom prospectively collected, contemporaneous clinical data including medication exposure were available.

Results: Compared to heathy controls, SLE patients showed over-expression of IFN and under-expression of B cell, T cell and pDC modules. Neutrophil module over-expression and under-expression of B and T cell modules were observed in patients with active lupus nephritis or highly active disease (SLEDAI-2K > 8), while Lupus Low Disease Activity State (LLDAS) had inverse associations. Disease activity in other organ domains was not associated with specific gene modules. In contrast, medications were associated with multiple effects. Glucocorticoid use was associated with under-expression of T cell, B cell and plasmablast modules, and over-expression of neutrophil modules. Mycophenolate and azathioprine exposure were associated with plasmablast module and B cell module under-expression respectively. Disease activity associations with neutrophil over-expression and lymphocyte module under-expression were attenuated by multivariable adjustment for medication exposure.

Conclusion: Medications have significant effect on gene module expression in SLE patients. These findings emphasize the need to control for medications in studies of gene expression in SLE.

Keywords: gene modules; glucocorticoids; immunosuppressants; systemic lupus erythematosus; transcriptional profiling.

Figure 1

Gene module expression in SLE patients compared to healthy controls depicted (A) as dot plots and (B) as relative log₂ fold change (log₂FC). Blue symbols represent modules that were significantly under-expressed, grey symbols modules that were not significantly different and red symbols represent modules that were significantly over-expressed in SLE patients compared with controls. Horizontal bars indicate 95% confidence intervals (95%CI).

Figure 2

Univariate analysis depicting log₂FC gene expression in SLE patients associated with (A) demographic factors, (B) disease manifestations at any time point in their documented disease course, (C) medication exposure at the time of sample collection, (D) SLEDAI-2K organ domain disease activity at time of sample collection and (E) composite disease activity measurements at the time of sample collection. * p<0.05 **p<0.01 ***p<0.001. Asian ancestry backgrounds are compared to European ancestry.

Figure 3

Gene module expression associations with disease activity (SLEDAI-2K) (expressed as log₂FC per SLEDAI point) in (A) univariate analysis, (B) analysis adjusted for prednisolone dose (pred), (C) analysis adjusted for prednisolone and mycophenolate (MMF) and (D) analysis adjusted for prednisolone and azathioprine (AZA) exposure. Blue symbols represent statistically significant under-expression of gene module, grey symbols represent results that were not significant, and red symbols represent statistically significant over-expression.

Figure 4

(A) Neutrophil module expression in patients with and without active renal disease and their associated prednisolone dose. (B) B cell module expression in patients with and without active renal disease and their associated prednisolone dose. (C) Expression of gene modules in patients with active renal disease compared to those without active renal disease in univariate analysis and (D) analysis adjusted for prednisolone exposure. (E) B cell module expression in patients with and without renal disease and their associated azathioprine use. (F) Expression of gene modules in patients with active renal disease compared to those without active renal disease adjusted for prednisolone and azathioprine (AZA) exposure and (G) prednisolone and mycophenolate (MMF) exposure.

Figure 5

Gene module expression in patients taking (A) rituximab, (B) belimumab and (C) cyclophosphamide. Grey dots represent samples from patients not taking these medications, colored dots represent samples taken from patients taking these medications. On each graph, each color represents samples from a single patient.