Background: 3BP2 is an adapter protein that mediates tyrosine kinase signaling. We have previously reported that 3BP2 is involved in cytokine production via Toll-like receptor (TLR) phosphorylation and innate immune regulation through Syk/SRC activation[1]. Although abnormal activation of tyrosine kinase/TLR pathways in immune cells is known to be involved in the pathogenesis of Systemic lupus erythematosus (SLE)[2], detailed mechanisms remain unclear.

Objectives: The objective of this study is to examine the role of 3BP2 in the pathogenesis of SLE and to provide the mechanism for 3BP2 as an optimal therapeutic target for SLE.

Methods: We generated B6/lpr CD72 knockout (KO) 3BP2KO mice by crossing CD72KO/lpr mice, which develop lupus nephritis due to abnormal B cell activation, with 3BP2KO mice. CD72 is a regulator of the B cell receptor (BCR) expressed mainly on B cells. B6/lpr CD72KO mice are a mouse model of lupus with a stronger phenotype than existing B6/lpr mice and can survive longer than MRL/lpr mice. In this study, we investigated the role of 3BP2 in the pathogenesis of lupus using these mice, with a particular focus on B cell signaling. First, histological evaluation of various organs, urinary protein determination, and plasma collection were performed in B6/lpr CD72KO and B6/lpr CD72KO 3BP2KO mice (CD72KO/3BP2KO). Flow cytometry (FCM) analysis was performed using peripheral blood mononuclear cells (PBMC) from spleen. Western blot and quantitative PCR were performed on B cells isolated from PBMC to analyze the effect of 3BP2 deficiency on intracellular signals.

Results: To determine whether 3BP2 is involved in the pathogenesis of SLE, we examined 3BP2 expression and found that 3BP2 was upregulated at the protein level in B cell, T cell and macrophage of lupus-prone mice compared to wild-type mice. Of note, observed a marked reduction of Alb/Cr ratio measured in urines collected from CD72KO/3BP2KO mice compared to CD72KO mice (CD72KO: 169.4 mg/gCr, CD72KO/3BP2KO: 23.98 mg/gCr, p value = 0.002). Inflammatory cell infiltration into lung and liver observed in CD72KO mice was also improved in CD72KO/3BP2KO mice. In addition, the infiltrating cells in glomeruli observed in CD72KO mice was improved in CD72KO/3BP2KO mice. FCM analysis of PBMC from CD72KO mice showed increased frequency of CD11c+/T-bet+ cells (Age or autoimmune-associated B cells (ABCs), that are involved in autoantibody production, while the frequency of ABCs was significantly decreased in PBMC from CD72KO/3BP2KO. In addition, the frequency of CD69-expressing active T cells was increased in PBMC from CD72KO mice compared to wild-type mice, while the frequency tended to be decreased in PBMC from CD72KO/3BP2KO. We next isolated B cells from spleen and examined B cell signaling and gene expression by stimulating BCR and TLR7 with IgM cross-linking and the TLR7 ligand ssRNA, respectively. We observed the increased level of Syk phosphorylation in B cells from CD72KO mice, that was decreased in CD72/3BP2-deleted B cells. In addition, we observed that the expression level of IL-6 mRNA was increased compared to ones from CD72KO stimulated by IgM and ssRNA, that was decreased in CD72KO/3BP2KO cells.

Conclusion: 3BP2 in highly expressed in the immune cells including B cells in SLE, and Syk phosphorylation and subsequent activation of the BCR and TLR7 signaling pathways. 3BP2 enhances differentiation of naive B cells into pathogenic ABCs, while deletion of 3BP2 can ameliorates proteinuria and systemic inflammation in lupus-prone mice. Our data provide new insights into the roles of 3BP2 for development of SLE and expand that concept that 3BP2 could be a therapeutic target for SLE.

REFERENCES: [1] Matsumoto Y, et al. J Clin Invest. 132(7): e140869, 2022.

[2] Kawahara K, et al. Internal J Molecular Science 17;22(8):4169, 2021.

Acknowledgements: NIL.

Disclosure of Interests: None declared.