Gene Expression Pathways across Multiple Tissues in Antineutrophil Cytoplasmic Antibody-associated Vasculitis Reveal Core Pathways of Disease Pathology

Abstract

Objective: To identify commonalities in gene expression data across all antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) tissues thus far characterized.

Methods: Gene expression data were collected from the 3 AAV tissues thus far characterized (orbit, peripheral leukocytes, and sinus brushings). These data were analyzed to identify commonly expressed genes and disease pathways. The pathways data were adjusted for multiple comparisons using a combined local false discovery rate, which estimates the probability of a false discovery of a given pathway in all 3 tissues analyzed.

Results: Only 4 genes were upregulated in all 3 tissues - IL1RN, TLR2, SLC11A1, and MMP9. After multiple comparison adjustments, the network pathway analysis revealed 28 pathways associated with all 3 tissues. The most strongly associated pathway for all 3 tissues was the neutrophil degranulation pathway [multidimensional local false discovery (md-locfdr) = 1.05 × 10^-12], followed by the osteoclast differentiation (md-locfdr = 3.8 × 10^-05), cell surface interactions at the vascular wall (md-locfdr = 4.2 × 10^-04), signaling by interleukins (md-locfdr = 6.1 × 10^-04), and phagosome (md-locfdr = 0.003) pathways. There were no downregulated genes or pathways common to all 3 tissues.

Conclusion: This analysis identified individual genes and pathways of disease common to all AAV tissues thus far characterized. The use of a network pathway analysis allowed us to identify pathologic mechanisms that were not readily apparent in the commonly expressed genes alone. Many of these pathways are consistent with current theories about infectious drivers and the crossroads of innate and adaptive immune mechanisms. In addition, this analysis highlights novel pathways, such as vessel wall interactions and platelet activation, which require further investigation.

Keywords: ANTINEUTROPHIL CYTOPLASMIC ANTIBODY–ASSOCIATED VASCULITIS; GENE EXPRESSION; GRANULOMATOSIS WITH POLYANGIITIS; METAANALYSIS; MICROSCOPIC POLYANGIITIS; VASCULITIS.

Figure 1:

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis pathophysiology revealed through gene expression pathways: 1. Infectious diseases, such as Staphylococcus aureus, activate Toll-like receptors (TLR) on antigen presenting cells and macrophages.(19) 2. Antimicrobial peptide homology with human antigens leads to the formation of pathogenic ANCAs.(20) 3. Macrophages secrete inflammatory cytokines which prime neutrophils— primed neutrophils express proteinase 3 (PR3) and myeloperoxidase (MPO) on their cell surface, allowing ANCA to bind and activate the neutrophil. 4. Cytokines and ANCAs activate endothelial cells, which express leukocyte adhesion molecules.(21-24) 5. ANCA-activated neutrophils migrate through the vascular wall where they degranulate, releasing reactive oxygen species and damage the vessel wall—thus causing the characteristic necrotizing vasculitis. 6. Platelets are activated at the sites of damaged endothelium, however their precise contribution to AAV pathophysiology is not well understood. 9. Activated neutrophils release neutrophil extracellular traps (NETs); NETs contain all of the intracellular contents of a neutrophil including MPO and PR3.(25) 8. Exposure of MPO and PR3 to the extracellular environment during NETosis drives further antibody formation. 9. Activated neutrophils also activate the alternative complement cascade, resulting in cleavage of C5 into C5a and C5b—C5a goes on to prime and attract more neutrophils leading to an amplification loop during active disease.(26)