Ann Dermatol.  2017 Apr;29(2):173-179. 10.5021/ad.2017.29.2.173.

Transcription Factors Regulating Inflammatory Cytokine Production Are Differentially Expressed in Peripheral Blood Mononuclear Cells of Behçet Disease Depending on Disease Activity

• Woo MY
• Yun SJ
• Lee MJ
• Kim K
• Lee ES
• Park S

• KMID: 2394840
• DOI: http://doi.org/10.5021/ad.2017.29.2.173

Abstract

BACKGROUND
Behçet disease (BD) is a relapsing inflammatory disease with increased production of inflammatory cytokines in peripheral blood mononuclear cells (PBMCs); however, the underlying molecular mechanisms are not well known.
OBJECTIVE
To analyze whether the differential expression of transcription factors is involved in the increased tumor necrosis factor (TNF)-α and interleukin (IL)-6 production by PBMCs of BD patients compared to healthy controls (HCs).
METHODS
Expression of transcription factors was examined by real-time reverse transcriptase-polymerase chain reaction and western blotting. Cytokine production by CD11b+ cells transfected with siRNAs against transcription factors was measured by enzyme-linked immunosorbent assay.
RESULTS
In the absence of lipopolysaccharide stimulation, the transcript level of CCAAT-enhancer-binding proteins (C/EBP) β was increased in PBMCs from patients with active BD compared to that in PBMCs from patients with stable BD. The C/EBPδ transcript level was higher in PBMCs from patients with active BD than in those from HCs. The activating transcription factor 3 (ATF3) transcript level was increased in PBMCs from patients with stable BD compared to that in PBMCs from HCs. siRNAs targeting C/EBPβ and C/EBPδ significantly reduced the production of IL-6 and TNF-α in lipopolysaccharide-stimulated CD11b+ cells from patients with BD as well as from HCs.
CONCLUSION
We found differential expression of C/EBPβ, C/EBPδ, and ATF3 in PBMCs from patients with BD depending on disease activity, indicating the involvement of these molecules in BD pathogenesis.

Keyword

Behcet syndrome; CCAAT-enhancer-binding proteins; Gene expression; Interleukin-6; Tumor necrosis factor-alpha

MeSH Terms

Activating Transcription Factor 3
Behcet Syndrome*
Blotting, Western
CCAAT-Enhancer-Binding Proteins
Cytokines
Enzyme-Linked Immunosorbent Assay
Gene Expression
Humans
Interleukin-6
Interleukins
RNA, Small Interfering
Transcription Factors*
Tumor Necrosis Factor-alpha
Activating Transcription Factor 3
CCAAT-Enhancer-Binding Proteins
Cytokines
Interleukin-6
Interleukins
RNA, Small Interfering
Transcription Factors
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 Increased lipopolysaccharide (LPS) concentration in sera of patients with Behçet disease (BD). LPS concentration was analyzed in the sera of five healthy controls (HCs) and 10 patients with BD (BD), using a Chromogenic LAL Endotoxin Assay Kit. The Mann-Whitney test was conducted. ***p<0.005.

• 

  Fig. 2 Differential mRNA expression of CCAAT-enhancer-binding proteins (C/EBP) β (A), C/EBPδ (B), and activating transcription factor 3 (ATF3) (C) in peripheral blood mononuclear cells (PBMCs) from Behçet disease (BD) patients. PBMCs isolated from 5 healthy controls (HCs), 4 to 6 stable BD patients (St) and 6 to 7 active BD patients (Ac) were cultured with or without lipopolysaccharide (LPS) for 3 hours. mRNA levels of C/EBPβ, C/EBPδ, and ATF3 were analyzed by real-time reverse transcription-polymerase chain reaction. Fold over HC(–): Relative mRNA level versus the average mRNA level in HC without LPS stimulation. Each symbol represents a single subject. Bars represent the mean of each group. The Kruskal-Wallis test with Dunn's procedure was conducted. *p <0.05, **p<0.01, ***p<0.005.

• 

  Fig. 3 Protein levels of CCAAT-enhancer-binding proteins (C/EBPβ), C/EBPδ, and activating transcription factor 3 (ATF3) in peripheral blood mononuclear cells (PBMCs) from Behçet disease (BD) patients. PBMCs isolated from healthy controls (HCs), stable BD patients (St), or active BD patients (Ac) were cultured with or without lipopolysaccharide (LPS) for 3 hours. Cell lysates were subjected to western blotting. Representative Western blots of nuclear lysates of 4 or 5 independent experiments (A). Relative band intensity to the indicated protein was compared between groups (B). Each symbol represents a subject and the bars represent the mean.

• 

  Fig. 4 Suppressive effect of siRNA targeting CCAAT-enhancer-binding proteins (C/EBP) β and C/EBPδ on the production of tumor necrosis factor (TNF)-α and interleukin (IL)-6. (A) Knockdown of the indicated transcription factors using specific siRNA. THP-1 cells were transfected with control siRNA (control) or siRNA specific for C/EBPβ, C/EBPδ, or ATF3. After 24 hours of transfection, protein levels were determined by Western blotting. un t/f: untransfected. (B, C) CD11b+ cells isolated from 5 healthy controls (HCs), 9 stable Behçet disease (BD) patients (St), and 10 active BD patients (Ac) were transfected with the indicated siRNA. After 24 hours, culture media was replaced with fresh media with or without lipopolysaccharide (LPS) (10 ng/ml). After 3 hours, the concentration of TNF-α and IL-6 in the media was measured. Relative production is the ratio of cytokine concentration in each culture condition relative to the average cytokine concentration in the control siRNA (con)-transfected culture without LPS stimulation. Each symbol represents a subject and the bars represent the mean of each group. Con, siRNA for C/EBPβ (siβ), siRNA for ATF3 (siATF3), a combination of siβ and C/EBPδ (siβ+δ). The Mann-Whitney test was conducted. *p<0.05, **p<0.01. un t/f: untransfection.

• 

  Fig. 5 Graphical summary. LPS: lipopolysccharide, ATF3: activating transcription factor 3, C/EBPβ LAP: CCAAT-enhancerbinding proteins β liver-enriched transcriptional-activator protein, HC: healthy controls, BD: Behcet disease, IL-6: interleukin-6, TNF-α: tumor necrosis factor-α.

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