Rheumηews

Presented By

Dr Walter Maksymowych MB ChB, FRCP(C), FACP
Chief Medical Officer, CARE Arthritis; Professor of Medicine, University of Alberta

Guest Commentary By:

Dr. Nigil Haroon MD, PhD
University Health Network

Synopsis

Introduction

An intergenic region on chr21q22 has been independently linked to inflammatory bowel disease, ankylosing spondylitis, primary sclerosing cholangitis and Takayasu’s arteritis (de Lange, K. M. et al., International Genetics of Ankylosing Spondylitis Consortium et al., Ji, S. G. et al., Ortiz-Fernandez, L. et al.)
Examination of H3K27ac chromatin immunoprecipitation–sequencing (ChIP–seq) data, which marks active enhancers and promoters, a monocyte/macrophage-specific enhancer has been identified within the locus
Using promoter-capture Hi-C and expression quantitative locus (eQTL) data from human monocytes (Methods), the disease-associated locus physically was shown to interact with the promoter of ETS2 and that the risk haplotype correlates with higher ETS2 expression
ETS2 is an ETS-family transcription factor and proto-oncogene, but its exact role in human macrophages is unclear, being reported as both pro- and anti-inflammatory (Wei, G. et al., Zhao, J. et al., Chung, S. W. et al., Quinn, S. R. et al., Ma, X. et al.)

Methods

Used a CRISPR–Cas9-based loss-of-function approach and designed two gRNAs targeting different ETS2 exons to assess impact of reducing expression of ETS2
Transfected non-activated macrophages with ETS2 mRNA and examined the impact of overexpression of ETS2exposure after stimulation with low dose lipopolysaccharide
Spatial transcriptomics and bulk RNA-seq data from diseased human tissues was used to examine ETS2 gene expression
Used MEK ½ inhibitors, which downregulate ETS-2 target genes, to assess anti-inflammatory activity in cultured macrophages and intestinal biopsies

Results

Single nucleotide polymorphism, rs2836882, in EST2 enhancer had strong EST2 gene expression-modulating effect
SNPs associated with ankylosing spondylitis were enriched in ETS2-target genes.
Targeting ETS2 markedly reduced pro-inflammatory cytokine production (IL-6, IL-8 and IL-1β), phagocytosis and oxidative burst, expression of inflammatory cytokines chemokines, secreted effector molecules, cell surface receptors, and signalling molecules
Overexpression of ETS2 resulted in dose-dependent macrophage activation of all inflammatory pathways
Differential expression of ETS2-regulated genes was observed in macrophages from IBD patients stratified by rs2836882 genotype
Transcriptional footprint of ETS2 was detectable in affected tissues from multiple chr21q22-associated diseases
MEK inhibition reduced inflammatory cytokine release to similar levels as infliximab
Several classes of drugs phenocopy ETS2 disruption, including JAK inhibitors.
Collectively, these data identify an essential role for ETS2 in macrophage inflammatory responses

Commentary

This landmark manuscript emphasizes the power of functional genomics to identify novel pathways mediating inflammation in axSpA and other immune-mediated disorders. An association of the intergenic region on chr21q22 with AS and IBD has been known for some time. This paper demonstrates that the region includes a polymorphism in an enhancer of the ETS2 gene that is present in most Europeans and Africans and has likely been conserved because of its essential role in activating macrophages in response to bacterial infection. Interestingly, blockade of ETS2 expression results in downregulation of proinflammatory genes also affected by JAK inhibitors though these agents do not directly affect expression of ETS2. A new target for therapeutic intervention in chronic inflammatory joint disease has been identified.

Special Guest Commentary

Following the use of GWAS, our understanding of genetic linkages to major inflammatory diseases including AS and IBD have significantly improved. This has also led to an explosion of functional genomics studies to help understand the pathological implications of these genetic associations. This study focuses on an intergenic region on chromosome 21q22, linked to several inflammatory conditions, and identifies the gene ETS2 as a key regulator of inflammatory macrophages. They then used the NIH LINCS database to identify drugs that might modulate ETS2 activity. There was a class effect of MEK inhibitors identified as strong modulators of ETS2 activity. Interestingly some benefit of using MEK inhibitors in pre-clinical colitis models has been previously shown. This work highlights the potential of functional genomics to elucidate disease mechanisms and uncover therapeutic avenues in immune-mediated disorders.