The efficacy of TNF antagonists and B cell depletion therapy (BCDT) in rheumatoid arthritis (RA) have fundamentally altered pathophysiologic paradigms and raised new questions about disease mechanisms. The observation that both TNF and B cells are of central importance provides a strong impetus to improve our understanding of the relationship between TNF over-production and inflammation pathways mediated by B cells, particularly in regards to disease flares and inadequate treatment response. The TNF-transgenic (TNF-tg) mouse strain is the only chronically progressive model for RA with a clinically proven etiology, and thus invaluable to address the cellular and immunological events in RA. We have developed a series of novel small animal imaging approaches, including contrast enhanced (CE) MRI, in vivo micro-CT and near-infrared indocyanin green (NIR-ICG) analysis of lymphatic flow, to study longitudinally the events associated with progression of joint arthritis. The results from these studies, combined with histology and flow cytometry, showed dramatic changes unfolding in a predictable temporal sequence in the draining popliteal lymph-node (PLN) before and during the onset of knee disease. Prior to knee synovitis, the draining PLNs "expand", as evidenced by increased volume and CE values (i.e. lower density), and histology of the node shows markedly dilated, enlarged sinusoids. This nodal expansion is also characterized by increased PLN B cell numbers, primarily due to accumulation of a B cell subset with a unique surface phenotype ("B-in" cells). This phase is followed by a rapid decrease in node size and CE, by invasion and "plugging" of the lymphatic sinuses by B cells, and reduced lymphatic flow through the node. The onset of knee synovitis and focal erosions follows PLN collapse. Both BCDT and anti-CXCL13 treatment result in TNF-tg disease amelioration and recovery of PLN size, CE and histology, pointing to a pathogenetic role of both B cells and chemotaxis. Thus, we hypothesize that evolution of knee arthritis and PLN function in TNF-tg mice proceed from an early stage during which pathogenetic B cells are recruited to and accumulate in the draining nodes, which increase in size and CE. At a later stage PLN-intrinsic or external (e.g. from the joint) signals induce migration of the expanded B cell population to the sinusoidal spaces, resulting in the obstruction of lymph flow and changes in the local cytokine balance. This leads to accumulation of pro-inflammatory mediators in the knee joint, triggering the "flare". Here we propose to test this model by: 1) identifying the mechanisms that lead to B cell accumulation in the PLNs and their subsequent migration to lymphatic spaces;2) demonstrating the pathogenetic role of adoptively transferred PLN B cells, and testing the hypothesis that BCDT efficacy is associated with clearance of sinusoid-invading B cells;3) testing whether TNF-tg B cells can exert pro-inflammatory effector functions;and d) investigating the applicability of these finding to RA patients in a pilot clinical trial in which PLN structure and lymphatic function will be assessed before and after BCDT, and correlated with clinical symptoms.

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Rheumatoid arthritis (RA) is a debilitating joint disease, which is manifested by episodic flares, and affects >1% of our populations. Here we propose to elucidate the role of B cells in arthritic flares, and evaluate a novel clinical outcome measures in RA patients undergoing B cell depletion therapy (rituximab) to better understand the differences between responders and non-responders.

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National Institute of Allergy and Infectious Diseases (NIAID)
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Emory University
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