Macrophage polarization toward M1 phenotype through NF-κB signaling in patients with Behçet’s disease

Background Macrophages are key innate immune cells implicated in the pathogenesis of Behçet’s disease (BD), and macrophage polarization plays a pivotal role in inflammatory response. This study aimed to investigate the role of BD serum on the phenotypes and functions of macrophage polarization. Methods BD or HC serum-treated human monocyte-derived macrophages (HMDMs) were examined M1/M2 phenotypes using flow cytometry and ELISA. The phagocytic capacity of HMDMs and CD4+T cell differentiation facilitated by HMDMs were measured by flow cytometry. Transcriptome analysis of BD and HC serum-stimulated HMDMs was conducted to identify differentially expressed genes. NF-κB signaling was examined using western blot to explore the mechanism of macrophage polarization induced by BD serum. Results BD serum-treated macrophages expressed a higher level of CD86, IL-12, and TNF-α and a lower level of CD163, which were compatible with the M1-like phenotype. Furthermore, BD serum-treated macrophages showed enhanced phagocytic capacity and promoted more Th1 cell differentiation. Sixty-one differentially expressed genes were identified between BD and HC serum-treated macrophages and were enriched in NF-κB signaling. BD serum-treated macrophages showed upregulated p-p65 and downregulated IκBα, and NF-κB inhibitor attenuated BD serum-stimulated M1-like phenotype. Conclusions BD serum promoted macrophage polarization toward a proinflammatory M1-like phenotype through NF-κB signaling and potentially facilitated inflammation in BD. M1 polarized macrophages may be a potential therapeutic target for BD. Supplementary Information The online version contains supplementary material available at 10.1186/s13075-022-02938-z.

and even life span, causing considerable financial costs to society and individuals. The pathogenesis of BD remains elusive, where genetic predisposition and environmental stressors might play together, leading to overactivation of the immune system and inflammatory damage of multisystems [4], mainly manifested by enhanced inflammatory responses and overexpression of proinflammatory cytokines [5]. Current therapies include glucocorticoids, immunosuppressive agents, and emerging TNF inhibitors. Therefore, an in-depth understanding of the pathogenesis and the development of new therapeutic targets are essential for improving the prognosis and reducing the disease burden of BD.
Macrophages are key innate immune cells that initiate adaptive immunity by antigen presentation and cytokine production. Macrophages play a critical role in inflammation through phagocytosis and the production of proinflammatory cytokines and chemokines. Macrophages are categorized into two subtypes: classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages). M1 macrophages are induced by lipopolysaccharides (LPS) and interferon-γ (IFNγ) and produce proinflammatory cytokines, while M2 macrophages are induced by IL4, IL-10, or IL-13 and exert anti-inflammatory and promote injury healing and tissue repair [6]. Macrophage polarization, defined as phenotypic and functional plasticity of macrophages, is potentially implicated in autoimmune diseases, such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren's syndrome (SS), and inflammatory bowel diseases (IBD) [7,8].
Recent studies suggest M1 macrophage polarization in BD [9]. TNF-α, IL-1β, IL-6, IL-8, and IL-12 are major proinflammatory cytokines secreted by M1 macrophages and are elevated in BD [10]. M1 macrophages are observed in herpes simplex virus (HSV)-induced BD mouse model [11], and BD serum induces healthy donor monocytes to polarize to M1 macrophage in vitro [12]. However, the potential function and mechanism of M1 macrophage polarization in BD remains largely unknown. GWAS study identifies CCR1 and IL10 as risk loci of BD, which might promote M1 macrophage [13]. In this study, we performed phenotypic and functional investigations on BD serum-induced M1 macrophage polarization. Furthermore, we performed transcriptome analysis on macrophages to explore potential mechanisms driving M1 activation of macrophages.

Patients and controls
Forty-five treatment-naïve active BD patients ( Table S1). All BD patients fulfilled the International Criteria for Behçet's Disease (ICBD) [14], and active BD was defined as Behçet's Disease Current Activity Form (BDCAF) score≥1 or with elevated erythrocyte sedimentation rate (ESR)/highsensitivity C-reactive protein (hsCRP). Forty-five genderand 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.
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.

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 stained cells were immediately analyzed on FAC-SAria 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) [15] 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) [16]. 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 [17], including GSE16385_UNTREATED_ 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.

Western blot
HMDMs were stimulated with BD serum or HC serum for 0, 15, 30, and 60 min. Total proteins of 1-2×10 6 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.

Statistical analysis
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).

BD serum promotes M1-like macrophage polarization
To explore the phenotype of BD serum-stimulated macrophages, we treated HMDMs with BD or HC serum as well as M0, M1, or M2 conditions. The phenotypes of macrophages are regulated by complex, dynamic environments, making it unlikely to define myeloid cell heterogeneity with a limited number of markers. Therefore, M1 and M2 macrophages were defined using CD86, CD163, CD206, IL-12, and TNF-α as previously described [20][21][22] Figure S2 and S3).

Transcriptome analysis of BD and HC serum-treated macrophages
To gain insights into the mechanism of BD seruminduced M1-like macrophage polarization, we next performed transcriptome analysis on macrophages stimulated with BD and HC serum (n=4). Principal component analysis (PCA) of the top 2000 variable genes showed distinct transcriptional patterns between two conditions (Fig. 3A). We identified 41 upregulated and 20 downregulated differentially expressed genes (DEGs, Fig. 3B,C and Supplemental Table S2), which showed a transcriptionally active gene signature in BD seruminduced macrophages. GO biological process and KEGG analysis revealed enriched migration and chemotaxisrelated gene sets (Fig. 3D), including upregulated CXCL1, CXCL2, CXCL3, CXCL5, and CCL13 (Fig. 3C). We validated that BD serum-treated macrophages produced more CXCL2 and CXCL3 (Supplemental Figure S7), which are characteristic of classical M1 macrophage activation [24]. KEGG and GSEA analysis showed enhanced inflammatory immune responses in BD serum-induced macrophages, including phagosome, IL-17 signaling pathway, and TNF-signaling pathway (Fig. 3D-F), which was confirmed by the phagocytosis test (Fig. 1E), enhanced Th17 differentiation (Supplemental Figure S6), and overproduction of TNF-α in BD serum-treated macrophages (Fig. 1D), respectively. A response to a molecule of bacterial origin (Fig. 3D) was implicated in BD seruminduced macrophages, supporting infections as a trigger of BD [25]. The enriched Nod-like receptor pathway and NF-κB pathway (Fig. 3D-F) suggested enhanced innate immune responses in BD serum-induced macrophages. Finally, we compared our gene signature with previous studies [18,19] and found it demonstrated an opposite pattern from M2 polarization conditions such as IL-4 and IL-10 stimulation (Fig. 3E). Taken together, the transcriptome analysis suggested that BD serum promoted macrophage polarization toward a proinflammatory M1-like phenotype.

Discussion
This study demonstrated the M1 polarization of macrophages induced by BD serum, with enhanced ability of phagocytosis and facilitating Th1 differentiation. We found NF-κB pathway activation through transcriptome analysis and confirmed its regulation on BD serum-stimulated M1 polarization. Imbalanced M1/M2 macrophage polarization has been reported in many autoimmune diseases, including SLE, RA, and IBD [7,8]. M1 phenotype and M1/M2 ratio are increased in an HSV-induced BD mouse model, and IL-4 treatment reduces the M1/M2 ratio and ameliorates the disease [11]. Alpsoy et al. report that M1 macrophage markers CD11c and CD64 are strongly expressed in macrophages maintained in BD serum [12]. We observed an M1-like profile in BD serum-treated macrophages with elevated CD86, IL-12, and TNF-α and reduced CD163, and an M0-like profile in HC serum-treated HMDMs. M1 macrophages are induced by LPS, IFNγ and (or) TNF-α, while M2 macrophages are induced by IL4, IL-10, or IL-13 [6]. We and other investigators [28][29][30] have confirmed that the levels of IFNγ and TNF-α, but not IL-10, were higher in BD serum. However, neutralizing IFNγ and (or) TNF-α did not abrogate M1 polarization induced by BD serum (data not shown). In addition, serum levels of LPS in BD patients are positively correlated with mucous disease activity [31], and the expression of LPS receptor TLR4 is higher in BD macrophages [32]. We speculated that higher levels of LPS and IFNγ or TNF-α in BD serum might orchestrate to induce M1-like polarization, which deserves to explore in future studies. Nevertheless, our study and others indicate that M1 macrophage polarization is a key mechanism of BD, and targeting M1 macrophages might be a therapeutic approach for BD. M1 macrophages exhibit higher phagocytic activity than M0 macrophages [33]. Accordingly, we found BD serum-stimulated macrophages showed enhanced phagocytotic ability through functional experiments and transcriptome analysis. Moreover, macrophages play an important role in the activation of the acquired immune response. In a chronic inflammatory context, M1 predominance and M2 insufficiency favor differentiation of T cell activation and differentiation [34,35]. IL-12 and IL-23 and (or) IL-1β, produced by activated macrophages, induce Th1 and Th17 differentiation, respectively [36,37]. IFNγ-producing Th1 cells and IL-17-producing Th17 cells are pathogenic in BD [10,38,39]. In this study, BD serum-treated macrophages upregulated IL-12 expression and promoted IFNγ + T-bet + and IL-17A + RORγt + CD4 + T cell differentiation, suggesting that BD serum-induced M1-like Transcriptome data revealed DEGs related to chemotaxis, including CXCL1-3, CXCL5, and CCL13 in BD serum-treated macrophages. CXCL1-3 and CXCL5 are expressed by M1 macrophages and control the recruitment of neutrophils during tissue inflammation [40][41][42], and BD is a systemic vasculitis featured by notable neutrophil infiltration [43], CXCL1-3 and CXCL5 might be the key chemokines produced by BD macrophages to overattract neutrophils. Additionally, CCL13 attracts monocytes, macrophages, and T cells [44,45]. These chemokine genes suggest the M1-like BD serum-induced macrophages promote chemotactic activity to other immune cells and play a role in the pathogenesis of BD.
We found that NF-κB signaling regulated M1 macrophage polarization induced by BD serum. NF-κB is an important prototypic signaling cascade that drives classical (M1) activation of macrophages [46]. After stimulation, IκBα is phosphorylated, ubiquitinated, and degraded in the cytosol via activated IKK. Subsequently, NF-κB transcription factors were translocated into the nucleus to initiate downstream effector mechanisms and induction of proinflammatory mediators such Fig. 4 NF-κB pathway mediated BD serum-treated M1-like macrophage polarization. A Representative western blot images (upper) and summary (lower) of NF-κB p65, phospho-p65 and IκBα of macrophages treated with BD (n=3) serum or HC (n=3) serum. Macrophages were pretreated with DHE and then were stimulated with BD serum for 48 h. B Representative histograms (left) and summary (right) of CD86 expression on DHE-treated and untreated macrophages stimulated with BD (n=6) serum and HC (n=6) serum. C TNF-α production by DHE-treated and untreated macrophages stimulated with BD serum (n=6) and HC (n=6) serum. Data were shown as mean±SD. *, p<0.05; **, p<0.01, ***, p<0.001, ****, p<0.001 by two-way ANOVA. M BD , BD serum-treated macrophages; M HC , HC serum-treated macrophages as TNF-α and IL-6 [47]. In addition, NF-κB is a central mediator of priming signal for nod-like receptor (NLR) inflammasome [48], which pathway was also enriched in our transcriptomic analysis. In the study, we observed the degradation of IκBα and phosphorylation of p65, as well as overexpression of TNF-α in BD serum-treated macrophages, which suggested NF-κB signaling activation. NF-κB inhibitor attenuated M1 polarization of macrophages stimulated by BD serum. NF-κB activation could be induced by and, in turn, result in an amplified inflammatory cytokine profile in BD, including TNF-α, IL-1β, IL-6, IL-8, and IL-12 [10,[49][50][51][52], the combined effect of which provides M1 polarization environment.
Glucocorticoids, which inhibit NF-κB signaling through induction of IκBα synthesis and inhibition of NF-κB activity, are effective for BD [53]. We inferred that glucocorticoids may suppress inflammation by inhibiting M1 macrophage polarization in BD patients. Thus, our study added a new layer of treatment mechanism of glucocorticoids, and targeting the NF-κB pathway might be a potential approach to reduce the inflammatory response of BD.

Conclusion
In summary, BD serum skews macrophage polarization toward the M1 phenotype by activating the NF-κB pathway, which shows enhanced phagocytosis and drives Th1 cell differentiation. Targeting abnormally polarized macrophages may be a potential therapeutic approach for BD.