Background RBP-J, a DNA-binding protein, serves as the central transcriptional regulator of the Notch signaling pathway. Prior in vitro work has demonstrated that RBP-J plays a critical role in macrophage activation, polarization, and modulating pro-inflammatory cytokine expression under LPS stimulation. Others have demonstrated that TNFa can induce myeloid derived suppressor cell (MDSC; suppressors of T cell function) activity via iNOS and arginase expression, both of which are strongly dependent on RBP-J and thus implicating its role in mediating potential downstream effects of MDSCs in inflammatory arthritis.
Objectives To evaluate the in vivo effects of RBP-J's conditional deletion using the Mx1Cre system, as well as in the myeloid cell compartment only, on pro-inflammatory cytokine expression, lymphoid tissue immunocyte composition, and MDSC populations, using a K/BxN serum transfer model of inflammatory arthritis.
Methods RBP-Jflox/flox Mx1Cre and LysM-Cre knock-out mice with controls (n=4-5 mice per 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 real-time PCR. Splenic tissue was pooled collectively from each group of mice for immunophenotyping through flow cytometry. The same was done for superficial inguinal and draining popliteal lymph node (LN) tissue, as well as bone marrow. Statistical analysis was done using the unpaired student's t-test with p<0.05 considered significant.
Results Mx1Cre deletion, but not myeloid-specific deletion, of RBP-J resulted in significantly heightened K/BxN serum-induced arthritis (up to 14 days post-induction) versus controls. Gene expression profiling of total joint tissue from RBP-J Mx1Cre deleted mice showed trends toward increased IL-12p40 expression, decreased IL-1b expression, with expression levels of IL-6, TNFa, Notch target genes, as well as bone turnover markers, being comparable. In contrast, myeloid-specific RBP-J deleted mice showed decreases in IL-12p40/IFNg gene expression with variable expression of IL-6, TNF, and Notch target genes. Post-arthritis induction, Mx1Cre deletion of RBP-J resulted in higher proportions of CD11b+ and Ly6G+ cells in splenic tissue. MDSC populations identified using combinations of CD11b/Ly6G/Ly6C/CD115 markers in lymphoid tissue and bone marrow demonstrated a downward shift in proportions with RBP-J deletion and TNFa expression.
Conclusions Deletion of RBP-J using the Mx1Cre system, but not myeloid-specific deletion, leads to heightened K/BxN serum-induced inflammatory arthritis. Along with selective modulation of pro-inflammatory cytokine gene expression in vivo, coupled with differences in myeloid cell composition and trafficking observed, including that of the MDSC population, this suggests that the stromal versus hematopoietic compartments expressing RBP-J may be contributing to these observations. Further functional studies to better characterize these conclusions are currently underway.
Acknowledgements This work was supported by a Rheumatology Research Foundation Scientist Development Award and NIH R01-AR056729.
Disclosure of Interest None declared