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Abstract
Objective
Granulomatosis with polyangiitis (GPA), formally known as Wegener's granulomatosis, is a systemic vasculitis with necrotizing granulomatous inflammation. As treatment directed against tumor necrosis factor (TNF)-α failed in GPA, we investigated whether “TNF-α signature” (i.e. gene expression profile of TNF-α activation) was present in peripheral blood mononuclear cells (PBMCs) of patients with GPA.
Methods
Gene expression profiling was performed using total RNA from PBMCs of 41 patients with GPA and 23 healthy control subjects using the Illumina microarray technique. Gene set enrichment analysis (GSEA) was performed to detect the presence of TNF-α signature using the curated list C2-V3.0 by the Broad Institute. False discovery rate＜0.05 was considered statistically significant.
Results
GSEA did not show significant enrichment of any TNF-α associated gene sets in GPA. Expression levels of genes upregulated by TNF-α did not differ between healthy control subjects, patients in remission (Birmingham Vasculitis Activity Score [BVAS]=0), and those with active disease (BVAS≥1). In addition, an unsupervised hierarchical clustering of those genes failed to cluster the samples into healthy control subjects and GPA in remission or with active disease. B cell activation signature was enriched in GPA patients.
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Figure 1.
Unsupervised hierarchical clustering of the TNF-α downstream genes failed to cluster the samples into healthy and GPA subjects. Expression pattern of TNF-α downstream genes of controls, inactive, and active GPA did not cluster. GPA: granulomatosis with polyangiitis, TNF: tumor necrosis factor.
Table 1.
Absence of TNF-α signature in patients with granulomatosis polyangiitis
| Gene set name | Description* | Gene set size | NES | Nominal p | FDR† q |
|---|---|---|---|---|---|
| A. Enriched in GPA | |||||
| TIAN_TNF_SIGNALING_NOT_VIA_ NFKB | Genes modulated in HeLa cells by TNF not via NFKB pathway | 15 | 1.65 | 0.016 | 0.049* |
| ZHANG_RESPONSE_TO_IKK_INHI BITOR_AND_TNF_UP | Genes upregulated in BxPC3 cells (pancreatic cancer) after treatment with TNFIKI-1, an inhibitor of IkappaB kinase | 158 | 1.26 | 0.052 | 0.255 |
| SANA_TNF_SIGNALING_UP | Genes upregulated in five primary endothelial cell types (lung, aortic, iliac, dermal, and colon) by TNF | 55 | 1.16 | 0.229 | 0.365 |
| SANA_TNF_SIGNALING_DN | Genes downregulated in five primary endothelial cell types (lung, aortic, iliac, dermal, and colon) by TNF | 58 | 0.91 | 0.622 | 0.750 |
| TIAN_TNF_SIGNALING_VIA_NFKB | Genes modulated in HeLa cells by TNF | 19 | 0.70 | 0.884 | 0.958 |
| BIOCARTA_TNFR1_PATHWAY | TNFR1 Signaling Pathway | 28 | 0.55 | 0.980 | 1.000 |
| B. Enriched in healthy controls | |||||
| BIOCARTA_TNFR2_PATHWAY | TNFR2 Signaling Pathway | 17 | 0.65 | 0.930 | 0.991 |
GSEA showed that only one of the downstream genes of TNF-α activation was enriched in patents with GPA as compared to healthy controls. FDR: false discovery rate, GPA: granulomatosis with polyangiitis, GSEA: gene set enrichment analysis, NES: normalized enrichment score, TNF: tumor necrosis factor, TNFR: tumor necrosis fator receptor.
* Description of gene sets are available on http://www.broadinstitute.org/gsea.
Table 2.
Presence of B-cell activation signature in granulomatosis polyangiitis
| Gene set name | Description* | Gene set size | NES | Nominal p | FDR† q |
|---|---|---|---|---|---|
| A. Enriched in GPA | |||||
| HUTTMANN_B_CLL_POOR_SURV IVAL_UP | Up-regulated genes in B-CLL patients expressing high levels of ZAP70 and CD38, which are associated with poor survival | 203 | 2.0 | ＜0.001 | 0.004 |
| FAELT_B_CLL_WITH_VH3_21_UP | Genes upregulated in samples from B-CLL with the immunoglobulin heavy chain VH3-21 gene | 39 | 1.71 | 0.009 | 0.038 |
| MOREAUX_MULTIPLE_MYELOMA _BY_TACI_UP | Up-regulated genes distinguishing in multiple myeloma samples with higher expression of TACI | 230 | 1.69 | 0.009 | 0.041 |
| TARTE_PLASMA_CELL_VS_PLASM ABLAST_UP | Genes upregulated in mature plasma cells compared with plasmablastic B lymphocytes | 255 | 1.73 | ＜0.001 | 0.031 |
| TARTE_PLASMA_CELL_VS_B_LYM PHOCYTE_UP | Genes upregulated in plasma cells compared with B lymphocytes | 74 | 1.68 | ＜0.001 | 0.043 |
| B. Not enriched in GPA | |||||
| SA_B_CELL_RECEPTOR_COMPLEX ES | Antigen binding to B cell receptors activates protein tyrosine kinases, such as the Src family, which ultimate activate MAP kinases | 22 | 1.26 | 0.168 | 0.257 |
| ST_B_CELL_ANTIGEN_RECEPTOR | B Cell Antigen Receptor | 37 | 1.03 | 0.406 | 0.557 |
| MOREAUX_B_LYMPHOCYTE_MAT URATION_BY_TACI_UP | Genes upregulated in normal bone marrow plasma cells compared to polyclonal plasmablasts that also distinguished multiple myeloma samples by expression of levels of TACI | 50 | 0.86 | 0.710 | 0.813 |
| C. Enriched in healthy controls | |||||
| HUTTMANN_B_CLL_POOR_SURV IVAL_DN | Down-regulated genes in B-CLL patients expressing high levels of ZAP70 and CD38, which are associated with poor survival | 49 | 2.25 | ＜0.001 | 0.010 |
| FAELT_B_CLL_WITH_VH_REARRA NGEMENTS_DN | Genes downregulated in B-CLL patients with mutated immunoglobulin variable heavy chain genes | 44 | 1.76 | 0.002 | 0.033 |
GSEA showed that the genes involved in B cell activation and survival were enriched in patents with GPA as compared to healthy controls. B-CLL: B-cell chronic lymphocytic leukemia, FDR: false discovery rate, GPA: granulomatosis with polyangiitis, GSEA: gene set enrichment analysis, NES: normalized enrichment score, MAP: mitogen-activated protein, TACI: transmembrane activator and calcium modulator and cyclophylin ligand interactor.
* Description of gene sets are available on http://www.broadinstitute.org/gsea.
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