Background/Purpose We recently showed that in SLE, IL-4 suppressed the development of interferon-beta (IFNβ) and TLR7-stimulated T-bet⁺ double negative 2 (DN2) B cells. Here we investigate the potential DN2 B-cell suppressive effects of IL-4 through the IL- 4-induced 1 (IL-4i1)-aryl-hydrocarbon receptor (AhR) pathway.

Methods The mechanism of IL-4 in suppressing the development of DN2 B cells in vivo was studied using R848-treated BXD2 mice. The B-cell developmental trajectory was determined using single-cell RNA-sequencing (scRNA-seq) analysis. Adult patients who met the ACR 1997 revised criteria for SLE were recruited. The in vitro effects of IL- 4 and two potent AhR agonistic ligands, Kynurenine (Kyn) and 6-formylindolo[3,2- b]carbazole (FICZ) in suppressing the development of DN2 B cell were determined using IFNβ plus TLR7 stimulated B cells. B-cell subsets and transcription factors (TFs) expression was measured by surface and intra-nuclear FACS analysis. AhR pathway and target genes were analyzed using qPCR. Autoantibodies were measured by ELISA.

Results Administration of IL-4 significantly inhibited the development of anti-Smith, anti-DNA, and anti-histone autoantibodies induced by the TLR7 agonist R848 in BXD2 mice. This was associated with a decreased percentage of CD11c⁺T-bet⁺ IgD⁻ B cells. Feature-barcoding single-cell RNA-sequencing analysis showed that IL-4 modulated B- cell development at the transitional stage 2 (T2) and skewed naïve B cells to develop into the CD23⁺CD21⁻ follicular B cells. IL-4 induced the gene encoding Ii4i1, an enzyme that metabolizes aromatic amino acids, and this was associated with the upregulation of AhR and downstream genes Cyp1a1 and Ido1. In the absence of IL-4, both Kyn and FICZ significantly suppressed TLR7 plus IFNβ-induced T-bet⁺ B-cell development in vitro in BXD2 mice. Analysis of AhR expression in healthy control (HC) and SLE subjects show a significantly lower expression of AhR in both the IgD⁻CD27⁻ DN and IgD⁺CD27⁻ naïve B-cell populations of SLE compared to HC. qPCR analysis further shows a significantly lower expression of CYP1A1 and IL4I1 in SLE B cells compared to HC B cells. B cell culture with either IL-4 or Kyn significantly reduced the development of DN2 B cells stimulated by IFNβ plus TLR7. Kyn also significantly induced the expression of CYP1A1 and promoted the expression of PD-1 in IFNβ plus TLR7- stimulated B cells.

Conclusion Our results suggest that IL-4R acts through the IL4i1-AhR pathway to induce a B-cell regulatory response to TLR7 and type I IFN. Identifying small molecular metabolites that act directly in B cells to induce homeostasis may lead to the development of orally active druggable targets that are efficacious in treating SLE.

This study was supported by a VA Merit Review grant (I01BX004049 and I01BX006099), NIH grants R01 AI134023, a Lupus Research Alliance Distinguished Innovator Award to JDM, an LRA Target Identification in Lupus Award to H-C.H., and the P30-AR-048311