Background/Purpose: The DNA-binding protein RBP-J serves as the central transcriptional regulator of the Notch signaling pathway. Prior work done using a knockdown approach of RBP-J in human macrophages and conditional deletion of RBP-J in mouse macrophages has demonstrated diminished LPS-induced expression of TNFa, IL-6 and IL-12p40, with IL-1 induction preserved. Elsewhere, it has been observed that host regulatory T cells in RBP-J deficient mice have an attenuated ability to suppress effector T cell responses in vitro, with augmented proliferation and function of effector T cells noted in vivo, raising the possibility that dysregulation in the frequency or function of regulatory T cells may contribute to RBP-J’s selective modulation of pro-inflammatory mediators. Here, we evaluated the in vivo effects of RBP-J’s conditional deletion in the myeloid cell compartment on pro-inflammatory cytokine expression, as well as lymphoid tissue immunocyte composition, using a K/BxN serum transfer model of inflammatory arthritis.

Methods: RBP-J^flox/flox LysM-Cre knock-out (KO) mice with littermate RBP-J^+/+LysM-Cre controls (n=5 for each group) were used. After treatment with K/BxN serum, the clinical course of arthritis was followed by measuring total joint thickness up to 14 days, at which point the mice were sacrificed. Total joint RNA from each mouse was obtained for gene expression analyses by qPCR. Splenic tissue was harvested from each mouse for gene expression analyses by qPCR, as well as pooled collectively for each group for immunophenotyping through flow cytometry. The latter was also done for superficial inguinal and draining popliteal lymph node (LN) tissue. Statistical analysis was done using the unpaired student’s t-test with p < 0.05 considered significant.

Results: Preliminary findings showed no significant difference in clinical phenotype of K/BxN serum-induced arthritis between KO and control mice. Gene expression profiling of whole joint tissue showed decreases in TNFa, IL-6, IFN-g expression, as well as selective Notch target gene expression, while maintaining comparable IL-1, CXCL10, and IL-12p40 levels. Surprisingly, expression levels of FoxP3 in KO mice vs. controls were significantly decreased in both joint and splenic tissue (p=0.0244 and p=0.0286, respectively). Immunophenotyping of splenic and LN tissue showed increased proportions of total CD4+ T cells in KO mice vs. controls, but a markedly lower proportion of the CD4+CD25+FoxP3+ subset. Lower proportions of F4/80+ and Ly6G+ cell populations in splenic and draining LN tissue of KO mice vs controls, but higher populations of Ly6C+ cells, were also observed.

Conclusion: Deletion of RBP-J in the myeloid compartment does not lead to phenotypic differences in K/BxN serum-induced inflammatory arthritis, though selective modulation of pro-inflammatory cytokine gene expression in vivo does occur. Decreased gene expression of FoxP3 and fewer CD4+CD25+FoxP3+ cells in RBP-J deleted mice may contribute to this selective modulation. The functional significance of these findings, coupled with differences in myeloid cell composition and trafficking observed, remain undetermined and will be further studied.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/deletion-of-rbp-j-in-a-murine-model-of-inflammatory-arthritis-reveals-differential-pro-inflammatory-cytokine-and-foxp3-gene-expression/