Date: Sunday, November 5, 2017
Session Title: B Cell Biology and Targets in Autoimmune Disease Poster
Session Type: ACR Poster Session A
Session Time: 9:00AM-11:00AM
Background/Purpose: Rheumatoid arthritis (RA) is a systemic autoimmune disease that often leads to joint damage, a process mediated by an imbalance between bone resorption and bone formation. B cells play a role in bone homeostasis in RA, and evidence suggests that B cells can affect function of both osteoblasts (OB) and osteoclasts (OC). In mouse models of RA, B cells were enriched in the subchondral bone marrow (BM) and synovium, and were adjacent to the bone surface. These B cells produce the inflammatory cytokines TNFα and CCL3, which drive dysregulation of bone homeostasis via their OB inhibitory and OC stimulatory effects. In vitro, synergistic production of these cytokines results from co-activation of the B cell receptor (BCR) and toll-like receptor 9 (TLR9). Therefore, we investigated the mechanisms underlying synergistic TNFα/CCL3 production by B cells in response to BCR and TLR9 co-activation.
Methods: Isolated B cells from peripheral blood of healthy controls (HC) were stimulated with 10 µg/mL anti-Ig (A+M+G) and CpG2006 for 4 hours, and production of TNFα and CCL3 was assessed by ELISA and qPCR. Multiple specific inhibitors were used to test the involvement of BCR, TLR9, and NF-κB signaling, as well as autophagy. Data is presented as mean±SEM. Statistical significance was determined by student’s t-test.
Results: Stimulation of B cells with α-Igs and CpG2006 induced significant production of TNFα and CCL3 compared to unstimulated B cells at mRNA and protein levels. Furthermore, cytokine production was significantly higher than levels from α-Igs or CpG2006 stimulations alone, suggesting a synergistic effect (CpG+α-Igs vs. CpG vs. α-Igs (ng/mL): TNFα: 2.71±0.42 vs. 0.33±0.04 (P<0.0001) vs. 0.70±0.23 (P=0.0008), CCL3: 3.69±1.00 vs. 0.77±0.29 (P=0.0031) vs. 0.64±0.18 (P=0.0037)). This synergistic production was dependent on TLR9 and BCR signaling, as blocking B cells with hydroxychloroquine (TLR inhibitor) or PP2 (Src kinase inhibitor) reduced cytokine production to non-synergistic levels. Additionally, blocking BCR internalization with MDC also reduced TNFα and CCL3 production. To investigate signaling downstream of BCR/TLR9 synergy, we tested inhibitors against classical NF-κB (TPCA-1), non-classical NF-κB (NIK inhibitor) and AP-1. We found that BCR/TLR9 synergy was classical NF-κB dependent (CpG+α-Igs vs. CpG+α-Igs+TPCA-1 (ng/mL): TNFα: 2.5±0.44 vs. 0.33±0.14 (P=0.009), CCL3: 1.9±0.3 vs. 0.83±0.08 (P=0.027)). Finally, we inhibited autophagy using class III PI3K inhibitor 3-methyladenine which reduced TNFα and CCL3 production to non-synergistic levels, suggesting that BCR/TLR9 synergy depends on autophagy (CpG+α-Igs vs. CpG+α-Igs+3-MA (ng/mL): TNFα: 1.5±0.25 vs. 0.4±0.04 (P=0.0058), CCL3: 4.1±1.15 vs. 0.82±0.35 (P=0.033)). Investigation of the specific autophagy pathway involved is currently underway.
Conclusion: Dual activation of B cells through the BCR and TLR9 synergistically enhanced TNFα and CCL3 production in a classical NF-κB, and autophagy-dependent manner. These results characterize key mechanistic events in the synergistic production of bone regulatory cytokines by B cells, a process that contributes to the dysregulation of bone homeostasis in RA.
To cite this abstract in AMA style:Glanzman J, Meednu N, Wang V, Sun W, Xing L, Anolik JH. Bone Regulatory Cytokine Production By B Cells in Rheumatoid Arthritis Is Dependent on NF-κb Activation and Autophagy Pathways [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/bone-regulatory-cytokine-production-by-b-cells-in-rheumatoid-arthritis-is-dependent-on-nf-%ce%bab-activation-and-autophagy-pathways/. Accessed September 18, 2021.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/bone-regulatory-cytokine-production-by-b-cells-in-rheumatoid-arthritis-is-dependent-on-nf-%ce%bab-activation-and-autophagy-pathways/