Background/Purpose: Interferon Regulatory Factors (IRFs) play fundamental roles in dendritic cell (DC) differentiation and function. In particular IRFs are critical transducers of Toll-like Receptors (TLR) signaling and their dysregulation is associated with the development of autoimmune disorders. While several IRFs are expressed in DCs, their relative contribution to the aberrant phenotypic and functional characteristics that DCs acquire in lupus has not been fully delineated. Mice deficient in DEF6 and SWAP-70 (=Double-knock-out or DKO mice), two members of a unique family of molecules that restrain IRF4 function, spontaneously develop systemic lupus erythematosus (SLE)-like disease in C57BL/6 mice. Although autoimmunity in DKO mice is accompanied by dysregulated IRF4 activity in both T and B cells, SWAP-70 is also known to regulate multiple aspects of DC biology. Here we thus explored whether DEF6 and SWAP-70 regulate IRF4 activity in DCs and investigated the contribution of IRF4 to DC dysfunction in the DKO mouse model.

Methods: CD11b⁺ and CD8⁺DCs were analyzed in vivo in WT and DKO mice by FACS. We investigated the expression of Blimp-1 and IL-10 in DCs in vivo by using a double reporter mouse model of these markers. Bone marrow derived DCs (BMDCs) were generated in vitro followed by TLR-4 and TLR-9 activation. BMDC activation markers, cytokine production and gene expression were analyzed by FACS, ELISA and qPCR. CD11c-Cre IRF4^fl/fl WT and DKO conditional knock out mice were also generated and analyzed in vivo and in vitro.

Results: We demonstrated that in vivo CD11b⁺ DCs in DKO mice are expanded and express higher levels of CD86 and PDL2 compared to WT mice. By using a dual reporter mouse model we determined that CD11b⁺DCs in DKO mice exhibit dysregulated IL-10 production, which is accompanied by aberrant Blimp1 expression in the spleen but not in skin draining lymph nodes. PDL2 expression was also upregulated in IL-10 producing DCs independently of Blimp-1 expression. Similar to our in vivo results, BMDCs from DKO mice show higher levels of CD86 and PDL2 compared to WT BMDCs and are hyper-responsive to multiple TLR ligands as assessed by their aberrant IL-10 and IFN-b expression. By using CD11c-Cre IRF4^fl/fl WT and DKO mice we also demonstrated that IRF4 plays a differential role in these responses by being required for the TLR4- but not the TLR9-mediated upregulation of IL-10. Finally in vivo deletion of IRF4 using the CD11c-Cre transgene led to a decrease in the CD11b⁺ DC population without any significant effects on the CD8⁺ DC subset. The lack of IRF4 did not significantly affect the upregulation of PDL2 and CD86 or the abnormal expansion of T_FH, Tregs, GC B cells and plasma cells that characterize DKO mice.

Conclusion: We demonstrated that CD11b⁺ DCs from DKO mice exhibit enhanced expression of Blimp1, PDL2 and dysregulated IL-10 production in vivo. Similar to other SLE mouse models, DCs from DKO mice were hyper-responsive to TLR stimulation. Deletion of IRF4 in DKO DCs in vivo and in vitro revealed that this phenotype is driven by an unexpected degree of molecular heterogeneity whereby lack of IRF4 corrected the TLR4-mediated hyper-responsiveness but exerted no effects on the TLR9-induced dysfunction.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/irf4-dependent-and-irf4-independent-pathways-contribute-to-dc-dysfunction-in-lupus/