The Oxysterol Receptor EBI2 Links Innate and Adaptive Immunity to Limit IFN Response and Systemic Lupus Erythematosus

Abstract Systemic lupus erythematosus (SLE) is a complex autoimmune disease with abnormal activation of the immune system. Recent attention is increasing about how aberrant lipid and cholesterol metabolism is linked together with type I interferon (IFN‐I) signaling in the regulation of the pathogenesis of SLE. Here, a metabonomic analysis is performed and increased plasma concentrations of oxysterols, especially 7α, 25‐dihydroxycholesterol (7α, 25‐OHC), are identified in SLE patients. The authors find that 7α, 25‐OHC binding to its receptor Epstein–Barr virus‐induced gene 2 (EBI2) in macrophages can suppress STAT activation and the production of IFN‐β, chemokines, and cytokines. Importantly, monocytes/macrophages from SLE patients and mice show significantly reduced EBI2 expression, which can be triggered by IFN‐γ produced in activated T cells. Previous findings suggest that EBI2 enhances immune cell migration. Opposite to this effect, the authors demonstrate that EBI2‐deficient macrophages produce higher levels of chemokines and cytokines, which recruits and activates myeloid cells,T and B lymphocytes to exacerbate tetramethylpentadecane‐induced SLE. Together, via sensing the oxysterol 7α, 25‐OHC, EBI2 in macrophages can modulate innate and adaptive immune responses, which may be used as a potential diagnostic marker and therapeutic target for SLE.


Supplementary materials and methods
The continuous variables were presented as the mean ± standard deviation.
Table S2.Detailed information on disease status at the time of blood collection, medication and treatment duration one month prior to blood collection for each SLE patients.Table S3.siRNA sequences for knockdown experiments.

Figure S2 .
Figure S2.The expression of EBI2 in monocytes of SLE patients or tissues of SLE mice.A) Heat map of DEGs in cMos of SLE patients with Asian ancestry versus those in HCs.B) The mRNA expression of IFNA and IFNB in PBMCs of healthy donors (n = 40) and SLE patients (n = 36).C) Flow cytometric analysis of monocytes in PBMCs of healthy donors (n = 24) and SLE patients (n = 24).D) scRNA-seq data of

Figure S3 .
Figure S3.Ebi2-cKO mice show normal macrophages development.A) The exon 2 in the Ebi2 gene was floxed to generate Ebi2 conditional knockout mice by CRISPR/Cas9 strategy.B-D) Ebi2 fl/fl mice were crossbred with Lyz2-Cre mice to delete EBI2 in myeloid cells (B).Ebi2 deletion was confirmed by genome sequencing (C), or in PEMs by qRT-PCR and immunoblot (D, n= 3).E-G) WT and Ebi2-cKO BMs were induced by M-CSF (20 ng/ml) for different days to detect the percentages of BMDMs in WT and Ebi2-cKO mice (E and F, n= 4), or to measure the mRNA levels of Adgre1 and Mertk (G, n= 3).Data are shown as mean ± SEM. **P < 0.01, using a two-tailed, unpaired Student's t test (D), or two-way ANOVA with Holm-Sidak's multiple comparisons test (F and G).

Figure S5 .
Figure S5.EBI2 does not affect the phosphorylation of TBK1 and IRF3.A) WT and Ebi2-cKO PEMs were stimulated with ISD to detect and quantify the phosphorylation levels of TBK1 and IRF3 (n = 3).B) WT and Ebi2-cKO PEMs were stimulated with ISD to quantify the phosphorylation levels of STAT1, STAT2, STAT3, ERK and p38 (n = 3).C) PEMs were pre-treated with 7α, 25-OHC followed by ISD stimulation to detect and quantify the phosphorylation levels of TBK1 and IRF3 (n = 3).D) PEMs were pre-treated with 7α, 25-OHC followed by ISD stimulation to

Figure S9 .
Figure S9.Analysis of peritoneal monocytes in TMPD-induced mice.A, B) Flow charts showing how to sort monocytes from peritoneal cells of TMPD-treated WT and Ebi2-cKO mice for RNA-seq.C) The expression of Ifit2, Ifi208, and Tlr9 in peritoneal monocytes of TMPD-treated WT (n = 5) and Ebi2-cKO mice (n = 5).D) Design of the transwell experiments.Data are shown as mean ± SEM. *P < 0.05 using a two-tailed, unpaired Student's t test (C).