Background Pathogenic extrafollicular double negative 2 (DN2) B cells in SLE have a phenotype of IgD⁻CD27⁻Tbet⁺CD11c⁺, and are the precursor of ribonuclear protein (RNP) autoantibody producing B cells. This trajectory of development is promoted by a synergistic effect of type I interferon (IFN) and TLR7-induced activation of B cells at the transitional (Tr) and naïve (NAV) developmental stages. In contrast, IL-4 and IL-4R expression is low in SLE patients and higher expression of IL-4 has been associated with a milder disease course in SLE. The present studies were carried out to determine the mechanism associated with IL-4- mediated B-cell quiescence program in vitro in SLE B cells and in vivo in the BXD2 mouse model of SLE.

Methods All SLE patients met the American College of Rheumatology 1997 revised criteria and the 2017 ACR/EULAR classification criteria for SLE. Peripheral blood mononuclear cells were analyzed by FACS for surface expression of IL-4R, intracellular IFN-β, and intranuclear T-bet and IRF7. DN2 B cell differentiation in vitro was stimulated with or without IL-4 50 ng/ml pre-culture for 1 hr. B-cell phenotypes, autoantibody profiles, and their association with the expression of IL-4R and IFN-β were analyzed in 47 SLE patients. The transcriptomics program associated with B-cell fate decision at the Tr, NAV, and activated naïve (aNAV) stages of B cell development in healthy control (HC) subjects and SLE subjects was analyzed using single cell RNA-sequencing (scRNA-seq) analysis. BXD2 mice were injected weekly with IL-4 complex or carrier anti-IL-4 antibody followed by R848, a TLR7 ligand. Four weeks later, mice were treated an additional IV injection of IL-4 complex or carrier. One week later, mice were sacrificed. The development of autoantibodies, GC B cells, DN2 B cells, and B-cell transcriptome were analyzed.

Results Section scRNA-seq analysis showed that type I interferon (IFN) stimulated genes (ISGs) were upregulated at Tr, rNAV, and aNAV stages of SLE B cells. In contrast, Tr and NAV B cells from SLE patients exhibited downregulation of an IL-4R quiescent gene program consisting of IL4R, BACH2, and FCRE2A (CD23). In HC, aNAV B cells exhibited upregulation of gene signatures of germinal center (GC) and classical memory (cMEM) B cells including LTB, GPR183, CD27, CD44, and CD83. IKAROS was identified as a top transcription factor associated with the upregulated genes in B cells from HC. In contrast, in SLE, aNAV B cells expressed signature genes of DN2 B cells including FCRL3, FCRL5, and ZEB2. Pseudotime analysis revealed in SLE, 63% of B cells developed into the DN2 pathway compared to only 3% in healthy controls. In contrast, only 14% of SLE B cells developed into the GC and classical memory pathway compared to 20% for healthy controls. IL-4 pre-treatment resulted in a significant increase of the CD11c⁻Tbet⁻ DN1 subpopulation and a decrease in the CD11c⁺Tbet⁺ DN2 B cells from SLE patients. There was a significant correlation of the percent of IL4R⁻IFNβ⁺ naïve B cells with SLEDAI, anti-Sm and anti-DNA. In vivo pre-treatment of BXD2 mice with IL-4 significantly blocked R848 induction of CD11c⁺CD21⁻Tbet⁺ DN2 B cells in the spleen. This was associated with a significant decrease in anti-DNA, anti-histone, anti-Sm, and anti-RNP autoantibodies. scRNA-seq analysis revealed that the molecular mechanism for IL-4 suppression of the R848 response was mediated through an transcriptome of aerobic metabolism and an IL-4-induced 1 (IL4i1)-Aryl hydrocarbon receptor (AhR) pathway. IL-4 inhibition of DN2 B cell development in human B cells in vitro was partially inhibited by the AhR inhibitor CH-223191.

Conclusion Low expression of the IL-4R program and low signaling through IL-4R at the Tr and naïve B cell stages in SLE pre-disposes such B cells to activation through TLR7. This can upregulate signaling through type II interferon and along with other stimuli to promote development of pathogenic DN2 B cells. Development of DN2 B cells can also be inhibited by IL-4R pathway agonist including treatment of cells with IL4i1 or other molecular activators of the AhR or tryptophan metabolites, such as IAA and IALD. Further studies of these pathways and molecules that can effectively and beneficially modify them could lead to improved treatment for SLE.

LAY ABSTRACT SLE is associated with excessive activation of lymphocytes, primarily B lymphocytes that can produce autoantibodies and pathogenic cytokines. Excessive B cell activation in SLE can be linked to several activation molecules including type I interferon, type II interferon, and cytokines including IL-17 and others. Much of the focus for treatment of SLE has been on neutralizing the pathogenic cytokines that drive the development of pathogenic B cells. In contrast, very little attention has been directed towards understanding how B cells in SLE patients become susceptible to activation through diverse pathways, and if lowered levels of immune mediators that maintain B lymphocyte in a quiescence status can contribute to the initiation of the autoimmune process. Previous studies are shown that while most of the inflammatory cytokines and factors are upregulated in SLE, IL-4 and its receptor (IL4R) are consistently downregulated in SLE patients. The present results have studied the effects of the low expression of IL-4R in the initial stages of B cell development in SLE. We have identified that IL-4R exhibited low expression whereas type I interferon exhibited high expression in SLE patients compared to normal controls. This imbalance in IL-4 and type I interferon resulted in a differential gene program regulation of B cells that prompts the activation and development of B cells into a pathogenic “DN2” population of B cells. In contrast, in normal controls, this IL-4R programed naïve B cell development promotes a different B-cell developmental trajectory that do not result in production of autoantibodies. The effects of IL-4 inhibiting pathogenic B cell development was analyzed in humans and in a mouse model of lupus. In both cases, pre- treatment with IL-4 blocked TLR7 and type I interferon-induced pathogenic “DN2” B cell development and suppressed the circulating levels of autoantibodies that are commonly seen in SLE patients. The molecular mechanism for IL-4R suppression of development of pathogenic B cells in lupus was found to be based on key molecules that regulate metabolism and quiescence of B cells. Further studies of these pathways and molecules that can effectively and beneficially modify them could lead to improved treatment for SLE.