ISR Autumn Meeting 2017

Basic Science Presentations - Joint Winner

Mary Canavan

TBA (17A174)

Joint Specific Transcriptional Programmes Regulate Inflammation in CD141+DC in Inflammatory Arthritis

Author(s)

Canavan M (1), Walsh, AM (2), McGarry T (1), Wade SM (1), Moran B (3), Biniecka M (4), Convery H (4), Wade S (1), Orr C (4), Mullan R (5), Fletcher JM (6), Nagpal S (2), Veale DJ (4), Fearon U (1)

Department(s)/Institutions

1. Molecular Rheumatology, School of Medicine, Trinity College Dublin, Ireland; 2. Immunology, Janssen Research & Development, 1400 McKean Road, Spring House, PA 19477, USA; 3. School of Biochemistry and Immunology, Trinity College Dublin, Ireland; 4. Centre for Arthritis & Rheumatic Diseases, Dublin Academic Medical Centre, University College Dublin, Ireland.; 5. Department of Rheumatology, Adelaide and Meath Hospital, Dublin, Ireland; 6. Schools of Biochemistry and Immunology and Medicine, Trinity College Dublin, Ireland.

Introduction

CD141+Dendritic Cells (DC) are implicated in anti-viral & anti-tumour immunity. However, due to their rarity in human blood and tissues, limited data exists on their role in autoimmune disease or rheumatic diseases.

Aims/Background

Our aims were to identify CD141+DC within the inflamed synovium of Inflammatory Arthritis (IA) patients, determine if synovial CD141+DC are distinct from peripheral blood (PB) CD141+DC and subsequently if this diversity translates into a joint specific phenotype.

Method

Synovial fluid/Peripheral mononuclear cells (SFMC/PBMC) were isolated from IA patients and CD141+DC were magnetically purified. Sorted cells were stimulated & stained with a panel of fluorochrome conjugated antibodies for multicolour flow cytometry. RNA sequencing was performed on CD141+DC from SFMC & PBMC and differentially expressed genes (DEG), Principal Component Analysis (PCA), Enriched pathway analysis, heatmaps and hierarchical clustering were identified using the DeSeq2 R package and Ingenuity® Pathway Analysis (IPA). For functional experiments, SF CD141+DC were cocultured with allogeneic CD3+T cells and intracellular cytokine quantified. Finally the effect of SF CD141+ DC-T cell cocultures on synovial fibroblast function was assessed.

Results

CD141+DC are significantly enriched in IA (SF) compared to blood and express XCR1, Clec9A and are negative for CD1c. RNASeq revealed 1417 upregulated genes in IA SF CD141+DC compared to PB CD141+DC & 1088 downregulated genes (FDR < 0.05; magnitude of fold-change > 2). Using hierarchical clustering and PCA analysis, SF CD141+DC clustered separately from PB CD141+DC, indicating the presence of distinct transcriptional variation between SF & PB CD141+DC. Specifically, we identified differential expression of genes involved in cell signalling (JAK2, JAK3, STAT2, STAT6), immune activation (CD53, CLEC9A, CD80) and cell adhesion (PLS3, ROBO, CD84) indicating the presence of unique gene signatures in synovial CD141+DC. To explore the functional consequence of this, DC–T cell coculture experiments were performed. SF CD141+DC induced proliferation of allogeneic CD4+ and CD8+T cells, with increased expression of IFNγ, TNFα, GM-CSF, IL-17A & Granzyme B. Furthermore SF CD141+DC-T cell interactions had the ability to further activate synovial fibroblasts, inducing IL-8, ICAM-1 and invasive mechanisms. Finally IPA identified joint-specific gene encoding pathways, including CD40, MAPK, and Jak-STAT, distinct to the synovial CD141+DC phenotype.

Conclusions

Synovial CD141+DC display unique mechanistic and transcriptomic signatures, contribute to synovial inflammation and are distinguishable from blood CD141+DC.