Patients and controls
Forty-five treatment-naïve active BD patients (27 males, age 33.0 ± 11.2 years, disease duration 94.4 ± 78.4 months) were recruited from Peking Union Medical College Hospital (PUMCH) between March 2014 and December 2019 (Supplemental Table S1). All BD patients fulfilled the International Criteria for Behçet’s Disease (ICBD) , and active BD was defined as Behçet’s Disease Current Activity Form (BDCAF) score≥1 or with elevated erythrocyte sedimentation rate (ESR)/high-sensitivity C-reactive protein (hsCRP). Forty-five gender-and age-matched healthy volunteers (25 males, mean age 36.8 years) were enrolled as healthy controls (HC). Serum samples of BD patients and paired HC were collected and stored at −80°C until use within 2 years. Twenty-nine paired BD and HC samples were used for the phenotype (n=12) and functional (n=17) analysis, 4 paired samples for bulk RNA-seq analysis, and the rest 12 paired for verifying the activated signaling pathway (Supplemental Table 1.1 and 1.2). The study was approved by the institutional review board of PUMCH, and written informed consent was obtained from all subjects in accordance with the Declaration of Helsinki.
Monocytes were isolated from HC peripheral blood mononuclear cells (PBMCs) with CD14+ MicroBeads (Miltenyi Biotec) with a purity >95% by flow cytometry (Supplemental Figure S1A). Monocytes (1.5×106 cells/mL) were seeded onto 24- or 48-well plates and were incubated in a complete DMEM medium supplemented with M-CSF (50ng/ml, Sigma) for 7 days to differentiate into adherent HMDMs. Complete DMEM contains DMEM (Gibco), 10% fetal bovine serum (FBS, Gibco), and penicillin and streptomycin (Gibco). The purity of HMDMs was > 80% measured by intracellular CD68 on day 7 (Supplemental Figure S1B). Naive CD4+ T cells were isolated from HC PBMCs using naive CD4+ T cell isolation kit II (Miltenyi Biotec) with a purity > 90% (Supplemental Figure S1C).
Resting (M0) macrophages were defined as HMDMs without additional stimulation. M0 were stimulated with LPS (100ng/ml) plus IFNγ (20ng/ml), or IL-4 (20ng/ml) plus IL13 (20ng/ml) for 48 h to differentiate into M1 or M2 macrophages, respectively. M0 were also treated with 10% BD or HC serum for 48 h. HMDMs were harvested using 0.25% trypsin-EDTA digestion for 10 min at 37°C. Surface CD86, CD163, and CD206 were measured with flow cytometry, and supernatant TNF-α, IL-12, CXCL2 and CXCL3 were measured by enzyme-linked immunosorbent assay (ELISA) (BioLegend or MultiSciences).
M0, M1, M2, and BD or HC serum-treated HMDMs were incubated in PBS with 1% BSA at 4°C for 30 min. After washing in PBS, cells were incubated with FITC-Dextran (Santa Cruz) and shaken at room temperature for 30 min. Cells were harvested and washed twice with PBS, 1% BSA. Intracellular FITC-dextran was determined by flow cytometry.
Macrophage-dependent T cell differentiation
M0, M1, and M2 conditions or BD and HC serum-pretreated HMDMs (5×104) were harvested and incubated with 2.5×105 naïve CD4+T cells in 48-well plates, and polarized in 500μl complete DMEM medium with plate-bound anti-CD3 (5 μg/ml, BD Biosciences), soluble anti-CD28 (5 μg/ml, BD Biosciences), anti-IL4 (5 μg/ml, BioLegend), and IL-2 (10 ng/ml, BioLegend) for 5 days. Before harvest, T cells were stimulated with Leukocyte Activation Cocktail (BD Bioscience) for 4 h, and IFNγ+ and T-bet+ CD4+ T cells were measured by flow cytometry.
For macrophages staining, macrophages were pretreated with Fc Receptor Blocking Solution (1:20, BioLegend) for 10 min at room temperature and were stained with surface antibodies and Ghost Dye (1:1000, Tonbo Biosciences) at 4°C for 30 min in dark. Macrophages were also fixed and permeabilized with Fixation/Permeabilization Solution (BD Biosciences) and were stained with anti-CD68 (Y1/82A, BioLegend). For T cell intracellular staining, cells were fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer (eBioscience), and intracellular cytokine/ nuclear transcription factor staining was performed according to the manufacturer’s protocol.
The following monoclonal antibodies (mAbs) were used: FITC anti-CD86 (BU63, BioLegend), PE anti-CD163 (GHI/61, BioLegend), APC anti-CD206 (15-2, BioLegend), and PerCP-Cy55 anti-CD68 (Y1/82A, BioLegend), FITC anti-CD4 (A161A1, BioLegend), PE-Cy7 anti-IFNγ (B27, BioLegend), PerCP-Cy55 anti-IL-17A (BL168, BioLegend), Alexa 647 anti-T-bet (O4-46, BD Biosciences), and PE anti-RORγt (AFKJS-9, BD Biosciences). Appropriately matched isotype control mAb to each antigen-specific mAb was used for control.
The stained cells were immediately analyzed on FACSAria II (BD Biosciences) flow cytometer, and data analysis was performed with the FlowJo software (Tree Star).
Bulk RNA-seq data analysis
Total RNA was extracted from BD or HC serum-treated HMDMs using TRIzol (Invitrogen). Sequencing libraries were generated with NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) and qualified by the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed using TruSeq PE Cluster Kit v3-cBot-HS (Illumia). The library preparations were sequenced on an Illumina Hiseq platform, and 125 bp paired-end reads were generated.
Raw data in FASTQ format were first processed through in-house Perl scripts. In addition, the Q20, Q30, and GC contents of the clean data were calculated. All downstream analyses were based on clean, high-quality data. A reference genome index was built, and paired-end clean reads were aligned to the reference genome using HISAT2 (v2.0.5). For quantification of gene expression levels, featureCounts (v1.5.0) was applied to count the number of reads mapped to each gene. The FPKM value of each gene was then calculated based on the length of the gene and the number of reads mapped to that gene.
The count matrix was input into DESeq2 (v1.30.0)  and fitted for a general linear model with a negative binomial distribution. To calculate DEGs, batch effect was corrected within DESeq2 and DEGs were identified by the functions DESeq with the adjusted p <0.05 (Wald test and Bonferroni correction). For PCA and heatmap demonstration, the matrix was corrected by R package sva to remove batch effect and normalized by Function rlog in DESeq2. PCA was performed for top 2000 variable genes based on variance, and the results were visualized with the function pca in R package PCAtools.
Pathway enrichment analysis
The enriched pathways were assessed by hypergeometric testing in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases based on DEGs by R package clusterProfiler (v3.0.4) . Significantly enriched pathways were determined with a cutoff of a Benjamini–Hochberg-corrected p < 0.05.
Gene signature analysis
Gene signatures were downloaded from the KEGG database and C7 gene sets of the MSigDB Collections , including GSE16385_UNTREATED_VS_12H_IL4_TREATED_MACROPHAGE_DN, GSE9509_LPS_VS_LPS_AND_IL10_STIM_IL10_KO_MACROPHAGE_30MIN_DN, and GSE25088_CTRL_VS_IL4_STIM_STAT6_KO_MACROPHAGE_UP gene sets from the previous studies [18, 19] which were relevant to our study. Significantly enriched pathways were determined with the cutoff of p value <0.05, Benjamini–Hochberg-corrected p < 0.25, and absolute value of negative normalized enrichment score (NES) >1.
HMDMs were stimulated with BD serum or HC serum for 0, 15, 30, and 60 min. Total proteins of 1–2×106 HMDMs were extracted with Minute Total Protein Extraction Kit (Invent Biotechnologies) and were quantified by BCA Assay Kit (Pierce). Proteins were loaded and electrophoresed on a 4–20% SDS-PAGE gel and were subsequently transferred to a PVDF membrane (Millipore). The membrane was blocked with tris-buffered saline-Tween 20 (TBST) containing 5% non-fat milk for 1 h at room temperature followed by incubation overnight with anti-NF-κB p65 rabbit antibody, anti-Phospho-NF-κB p65 rabbit antibody, anti-IκBα rabbit antibody, anti-JAK1 mouse antibody, anti-Phospho-JAK1 rabbit antibody, anti-STAT1 rabbit antibody, anti-Phospho-STAT1 rabbit antibody or anti-β-actin rabbit antibody (Cell Signaling Technology) at 4°C. The membrane was washed three times and incubated with HRP-conjugated secondary antibodies (EASYBIO) for 1 h at room temperature. The proteins were visualized using a ChampChemi Multiplex Fluorescence /Chemiluminescence Imager (Sage Creation Science), and the optical density data were analyzed using ImageJ software. β-actin was used as the endogenous control.
Quantitative data were expressed as mean ± standard deviation (SD) or median (range). Categorical variables were represented as frequencies and percentages. Student’s t test was used for comparing two groups. Multiple group comparisons were analyzed using one-way ANOVA and two-way ANOVA (normally distributed data) or Kruskal-Wallis test (non-normally distributed data). A two-sided p value < 0.05 was considered statistically significant. Analyses were performed with SPSS V.26 (SPSS, USA).