High-throughput RNA sequencing reveals distinct gene signatures in active IgG4-related disease

Abstract

We aimed to characterize the molecular differences and effects from prednisone treatment among IgG4-related disease with salivary gland lesions (RD-SG), without SG lesions (RD-nonSG), and IgG4-related retroperitoneal fibrosis (RF). RNA sequencing was conducted on blood from 25 RD-SG, 11 RD-nonSG, 3 RF and 10 control subjects. Among these, 8 RD-nonSG and 12 RD-SG patients were subjected to treatment with prednisone and/or glucocorticoid-sparing agents. Six RD patients had a longitudinal time point. The mRNA levels of IgG4 and IgE, genes specific for Th2 cells, eosinophils, and neutrophils were over-expressed in RD-SG and RD-nonSG. A B-cell signature was suppressed in patients group versus controls, while Th1, Th2, Treg, and eosinophil gene signatures were increased in patients without treatment. Interestingly, Tfh genes and B cell signature were decreased at flare disease state. Prednisone treatment led to increased neutrophil, but decreased Treg signatures. Serum IgG4 levels correlated with the eosinophil and neutrophil gene signatures in RD-SG patients, and with a B cell signature in only RD-nonSG patients. IgG4, IgE, and cell-specific signatures are regulated in patients, suggesting the imbalance of immune and inflammatory cells in IgG4-related disease. Prednisone treatment selectively modulates Treg, eosinophil, and neutrophil signatures.

Figure 1

Principal components analysis plot of RD-SG, RD-nonSG, RF, and control subjects using the whole transcriptome.

Figure 2

Distribution of IgG4 and IgE mRNAs. (A) Expression of IgG4 scaled by all IgG1, IgG2, and IgG3 transcripts and (B) IgE across control subjects, RD-SG patients on prednisone treatment, RD-SG patients not on prednisone treatment, RD-nonSG patients on prednisone treatment, RD-nonSG patients not on prednisone treatment, RF patients, all patients on predisone treatment, and all patients not on prednisone treatment. (C) Correlation between IgE and IgG4/Total IgG mRNAs for all three diseases. P-values under each disease group indicate comparisons to control and are adjusted by age. Pred +  = currently treated with prednisone; Pred− = not currently treated with prednisone.

Figure 3

Distribution of cell-specific gene signatures. Expression of gene signatures of (A) Treg, (B) IL-13, (C) IL-4, (D) B cell, (E) eosinophil, and (F) neutrophil across control subjects, RD-SG patients on prednisone, RD-SG patients not on prednisone, RD-nonSG patients on prednisone, RD-nonSG patients not on prednisone, RF patients, all patients on prednisone combined, and all patients not on prednisone. P-values under each disease group indicate comparisons to control and are adjusted by age. Pred +  = currently treated with prednisone; Pred− = not currently treated with prednisone.

Figure 4

Heatmap of cell-specific gene signatures across the control, RD-SG, RD-nonSG, and RF cohorts. Ctrl = control subjects; Pred +  = currently treated with prednisone.

Figure 5

Longitudinal gene or gene signature profiles of two patients at relative baseline and post relative baseline visits when a flare was observed. All plots include the control cohort and all the 6 patients for (A) IgG4/Total IgG, (B) IgE, (C) eosinophil gene signature, (D) Th1 gene signature, (E) Th2 gene signature, (F) Treg gene signature, (G) BCL6, (H) CXCR5, and (I) B cell gene signature. Pink = patient experienced flare at visit 2; Blue = patient had stable disease at visit 2.

Figure 6

Correlation between serum levels of IgG4 and (A) Treg signature, (B) IL13 signature, (C) IL4 signature, (D) B cell signature, (E) eosinophil signature, (F) neutrophil signature in RD-SG, RD-nonSG, and RF patients.