Deciphering Pathways Driving Inflammatory Fibroblast Effector Functions in RA

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In rheumatoid arthritis (RA), joint destruction is mediated by the synovium, which becomes inflamed, hyperplastic, and infiltrated by leukocytes. Fibroblasts constitute the synovial lining and the stromal network of the sublining layer. Current therapies for RA reduce disease activity but rarely achieve remission. Combinations of immunosuppressive biologics result in increased infectious complications limiting their use. We and others are working toward a distinct approach, namely to target synovial fibroblasts and/or their distinct pathways to abrogate inflammation independent of targeting leukocytes or inflammatory cytokines. In a recently published report (Nguyen et al. Immunity 2017) and preliminary data, we illustrate how cytokines like TNF, IL-1b and IL-17 (primary signals) all can activate fibroblasts to produce an array of inflammatory cytokines (e.g. IL-6) and chemokines (e.g. CCL2, IL-8) and growth factors (e.g. G-CSF) that highlight the role of fibroblasts themselves as inflammatory cells in RA. Importantly, we found that to achieve strong and sustained expression of these inflammatory cytokines, chemokines and growth factors, a critical amplification loop (secondary signal) is essential. This secondary signal is an autocrine positive feedback loop dependent on signaling downstream of the leukemia inhibitory factor receptor (LIFR). Silencing this receptor abrogates fibroblast production of IL-6 and a set of co-expressed inflammatory mediators, regardless of which primary signal is used to activate the fibroblasts. We hypothesize that the LIFR amplification loop is a critical shared component of fibroblast activation that may be targeted to abrogate fibroblast mediated inflammation in RA. In Aim 1, we define the LIFR positive feedback amplification loop signature by RNA sequencing after stimulating fibroblasts with a series of potent activators including, TNF, IL-1b, IL-17 and LPS. In Aim 2, we validate the key genes and products that make up the LIFR positive feedback loop signature using RT-PCR and protein determinations by ELISA and flow cytometry. To validate key effector functions, we examine the effects of LIFR deletion or silencing in in vitro functional assays that measure leukocyte recruitment and survival and other functions. In Aim 3, we assess LIFR amplification loop activity in active human RA and in TNF and methotrexate inadequate responders to assess if this pathway is a component of ongoing inflammation in RA. Finally, in Aim 4, we determine if targeting the LIFR amplification loop can be an effective therapy for inflammatory arthritis in mouse models. Together, these studies provide mechanistic insights into the pathways utilized by fibroblasts that drive inflammation in RA with implications for treatment.