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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus

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.
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Figure 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.

Mentions: We then evaluated the relevance of differential mRNA expression of C/EBPβ, C/EBPδ, and ATF3 to the increased production of TNF-α and IL-6 in CD11b+ cells of active BD using siRNA. First, we confirmed the successful knockdown of these transcription factors in THP-1 cells transfected with siRNA against each gene, comparing to protein levels observed in non-transfected cells or cells transfected with an unrelated, control siRNA (Fig. 4A). We next transfected CD11b+ cells with siRNAs targeting ATF3 or C/EBPβ alone or for both C/EBPβ and C/EBPδ. However, we could not include the transfection condition of siRNA targeting C/EBPδ alone due to the limited number of CD11b+ cells from each subject. After 24 hours, we transferred cells into fresh media with or without LPS, and 3 hours later assessed the amount of TNF-α and IL-6 in the media (Fig. 4B, C). In PBMCs of stable BD, LPS-induced production of TNF-α and IL-6 was significantly suppressed through transfection of siRNA targeting C/EBPβ alone or C/EBPβ in combination with C/EBPδ, using an equal amount of siRNA mixture for each condition (p<.0.05). Similarly, significant suppression of cytokine production by the transfection of siRNA targeting C/EBPβ alone or C/EBPβ in combination with C/EBPδ was observed in cells of patients with active BD, although modulation of TNF-α by siRNA targeting C/EBPβ alone was not statistically significant. Taken together, these results demonstrate that C/EBPβ and C/EBPδ contribute to the production of TNF-α and IL-6 in PBMCs from patients with BD.


Transcription Factors Regulating Inflammatory Cytokine Production Are Differentially Expressed in Peripheral Blood Mononuclear Cells of Beh ç et Disease Depending on Disease Activity
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.
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Related In: Results  -  Collection

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Figure 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.
Mentions: We then evaluated the relevance of differential mRNA expression of C/EBPβ, C/EBPδ, and ATF3 to the increased production of TNF-α and IL-6 in CD11b+ cells of active BD using siRNA. First, we confirmed the successful knockdown of these transcription factors in THP-1 cells transfected with siRNA against each gene, comparing to protein levels observed in non-transfected cells or cells transfected with an unrelated, control siRNA (Fig. 4A). We next transfected CD11b+ cells with siRNAs targeting ATF3 or C/EBPβ alone or for both C/EBPβ and C/EBPδ. However, we could not include the transfection condition of siRNA targeting C/EBPδ alone due to the limited number of CD11b+ cells from each subject. After 24 hours, we transferred cells into fresh media with or without LPS, and 3 hours later assessed the amount of TNF-α and IL-6 in the media (Fig. 4B, C). In PBMCs of stable BD, LPS-induced production of TNF-α and IL-6 was significantly suppressed through transfection of siRNA targeting C/EBPβ alone or C/EBPβ in combination with C/EBPδ, using an equal amount of siRNA mixture for each condition (p<.0.05). Similarly, significant suppression of cytokine production by the transfection of siRNA targeting C/EBPβ alone or C/EBPβ in combination with C/EBPδ was observed in cells of patients with active BD, although modulation of TNF-α by siRNA targeting C/EBPβ alone was not statistically significant. Taken together, these results demonstrate that C/EBPβ and C/EBPδ contribute to the production of TNF-α and IL-6 in PBMCs from patients with BD.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Beh&ccedil;et 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)-&alpha; 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) &beta; was increased in PBMCs from patients with active BD compared to that in PBMCs from patients with stable BD. The C/EBP&delta; 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/EBP&beta; and C/EBP&delta; significantly reduced the production of IL-6 and TNF-&alpha; in lipopolysaccharide-stimulated CD11b+ cells from patients with BD as well as from HCs.

Conclusion: We found differential expression of C/EBP&beta;, C/EBP&delta;, and ATF3 in PBMCs from patients with BD depending on disease activity, indicating the involvement of these molecules in BD pathogenesis.

No MeSH data available.


Related in: MedlinePlus