OP0296 Hypoxia-Inducible Factor 2A Regulates Macrophage Function in Rheumatoid Arthritis

Abstract

Background In rheumatoid arthritis (RA), the influx of inflammatory cells as well as the aggressive proliferation of fibroblast-like synovial cells (FLS) outstrips the oxygen supply from blood vessels leading to joint hypoxia. Macrophages accumulate in these hypoxic sites where they possess broad pro-inflammatory, destructive and remodelling potential leading to inflammation and joint destruction. Macrophages respond to such hypoxia by up regulating the hypoxia inducible transcription factors – HIF-1a and -2a, which leads to the up-regulation of genes involved in proliferation, angiogenesis, and glucose metabolism. Objectives To characterise the HIF-2a expressing macrophage populations in response to joint hypoxia, and in particular to dissect out the effects of HIF-2 in a murine model of arthritis. Methods Tissue from RA patients was assessed for oxygen levels using an oxygen/temperature probe then sections were immunostained with anti-HIF-2a and co-localised with the pan-macrophage markers CD68 as well as other M1 and M2 macrophage markers. Cell-specific RNA was purified from CD68+ macrophages in RA tissue by laser capture microdissection and differential gene expression was determined using the RT2 Profiler PCR inflammatory arrays. The significance of HIF-2a was also evaluated in the K/BxN serum transfer model in mice bearing a targeted deletion of HIF-2a in myeloid cells including macrophages (HIF2fl/fl; LysM-Cre+' mice). Arthritis was assessed clinically and histologically. Results In patients with severely hypoxic tissue (pO2<20 mmHg), CD68+ macrophages predominately expressed HIF-2a compared to macrophages from mildly hypoxic (pO2>20 mmHg) tissue (58%±10.2 vs. 15±9.3 p=0.01, respectively). This reduced in vivo oxygen tension (pO2<20 mmHg) also positively correlated with macrophages expressing GLUT 1 and receptors for VEGF (Flt1), angiopoietin “Tie2” and mannose “CD206”, all markers associated with M2-skewed macrophages. In addition, these hypoxic macrophages expressed increased mRNA for CXCL-2, -4, -5, IL-1a, IL-6, IL-8 & TNFa compared to macrophages from mildly hypoxic joints. In vivo, macrophages lacking HIF-2a significantly suppressed disease development indicating an indispensable role for HIF-2 in supporting macrophages in K/BxN serum-transfer arthritis. Conclusions HIF-2a expressing macrophages have an M2-like phenotype in patients with severely hypoxic joints and loss of HIF-2a in macrophages suppressed arthritis in mice. Collectively, our data identify HIF-2a as an important regulator of macrophage function, suggesting it may be a useful therapeutic target for treating RA. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4257

Hypoxia-Inducible Factor (HIF) as a Target for Novel Therapies in Rheumatoid Arthritis

“…In particular, HIF-2α expression was mainly observed in FLS of the RA synovium (Ryu et al, 2014 ). Furthermore, HIF-1α and HIF-2α have also been identified in resident and infiltrating immune cells (e.g., macrophages, T cell, and mast cells), chondrocytes, and osteoclasts (Westra et al, 2010 ; Hardy et al, 2014 ; Ryu et al, 2014 ). Understanding the expression of HIFs within the RA joint allows us to better understand the degree in which they are activated based on disease severity, as well as how they affect particular cell types to contribute to perpetuating the disease.…”

“…Immune cells also play an important role in synovial inflammation. HIF-1α and HIF-2α are highly expressed in immune cells, in particular macrophages, in the RA synovium (Hollander et al, 2001 ; Brouwer et al, 2009 ; Hardy et al, 2014 ). Resident macrophages (Type A synoviocytes) are primarily located in the intimal lining of the RA synovium, and are also found in the sub-lining layer and at the cartilage pannus junction (Kinne et al, 2000 ; Szekanecz and Koch, 2007 ).…”

“…Resident macrophages (Type A synoviocytes) are primarily located in the intimal lining of the RA synovium, and are also found in the sub-lining layer and at the cartilage pannus junction (Kinne et al, 2000 ; Szekanecz and Koch, 2007 ). In response to hypoxia, macrophages upregulate HIFs to activate pathways involved in proliferation, angiogenesis, and glucose metabolism (Hollander et al, 2001 ; Brouwer et al, 2009 ; Hardy et al, 2014 ). Cramer et al reported a significant reduction in synovitis, pannus formation, and disease progression with HIF-1α knockout myeloid cells in the K/BxN serum transfer arthritis model (Cramer et al, 2003 ).…”

“…Deletion of HIF-1α in myeloid cells reduced disease severity in both acute and chronic inflammation. Similarly, macrophages lacking HIF-2α significantly suppressed disease development in the K/BxN serum transfer arthritis model, indicating an important role for HIF-2α in supporting macrophages (Hardy et al, 2014 ).…”