Hypoxia Resistant Pathogenic B cells Accumulate in the RA Synovial Tissue in a CXCR3 Dependent Manner.
Achilleas Floudas1, Candice Low2, Monika Biniecka1, Murray Kierran2, Mullan RH3, Douglas J. Veale2, Ursula Fearon1
1Molecular Rheumatology, Trinity Biomedical Sciences Institute, TCD 2EULAR Centre of excellence, Centre for Arthritis and Rheumatic Diseases, St Vincent’s University Hospital, UCD. 3Dept of Rheumatology, Tallaght University Hospital, TCD
Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease of unknown and complex aetiology with severe detrimental effects for the patient’s quality of life. While autoantibodies have been used extensively for the diagnosis of RA, no clear mechanism of action towards disease pathogenesis and progression has been identified.
Identification of chemokines and their receptors responsible for the accumulation of invading B-cells in the inflamed joint.
Characterization of B-cell function under the unique hypoxic conditions of the RA joint.
Synovial tissue biopsies from RA patients with paired blood/synovial fluid, were obtained through key-hole arthroscopy followed flow cytometric analysis of B-cell subpopulations and chemokine receptor expression in the periphery and synovial tissue. Multiparametric SPICE analysis for the chemokine receptor expression (CXCR5, CXCR3, CCR7, CCR6. CCR4) of synovial tissue invading B-cells was performed followed by B-cell invasion assays. Functional characterization of RA sorted B-cells, cultured in vitro in a hypoxia chamber simulating the unique environment of the inflamed joint.
There is a significant accumulation of CD27+ and double negative (CD27-IgD-) memory B-cells in the synovial tissue and synovial fluid of RA patients irrespective of ACPA status. SPICE analysis of peripheral blood B-cells for a panel of chemokine receptors revealed a definitive bias towards a specific disease dependent chemokine receptor expression pattern, present from arthralgia-early in disease subjects. Tissue invading B-cells showed a clear preference for the expression of CXCR3. . Importantly returning RA patient B-cells following rituximab mediated B-cell depletion express high levels of CXCR3. By simulating the unique hypoxic conditions of the inflamed joint, we observed significant alteration in B-cell activation with RA B-cells showing increased pro-inflammatory cytokine production and Glut1 expression. B-cell Glut1 expression correlates with pSTAT3, while blockade of glucose uptake by 2DG abolishes RA B-cell pro-inflammatory cytokine production under normoxic or hypoxic conditions.
The accumulation of pro-inflammatory B-cell subpopulations in the synovium of RA patients, is CXCR3 mediated and offers an opportunity for early therapeutic intervention. Once in the hypoxic environment of the inflamed RA joint, B-cells show altered activation, pro-inflammatory cytokine production and metabolism that could prove important for understanding the role of B-cells in disease pathogenesis of RA.