Enhanced interferon regulatory factor 3 binding to the interleukin-23p19 promoter correlates with enhanced interleukin-23 expression in systemic lupus erythematosus

Siobhán Smith, Joan Ní Gabhann, Rowan Higgs, Kevin Stacey, Marie Wahren-Herlenius, Alexander Espinosa, Maria Grazia Totaro, Antonio Sica, Elizabeth Ball, Aubrey Bell, James Johnston, Peter Browne, Lorraine O'Neill, Grainne Kearns, Caroline A. Jefferies

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Abstract

Objective To examine the role of interferon regulatory factor 3 (IRF-3) in the regulation of interleukin-23 (IL-23) production in patients with systemic lupus erythematosus (SLE). Methods Bone marrow-derived macrophages were isolated from both wild-type and IRF3 -/- C57BL/6 mice. These cells were stimulated with the Toll-like receptor 3 (TLR-3) agonist poly(I-C), and IL-23p19 cytokine levels were analyzed by enzyme-linked immunosorbent assay. IRF-3 binding to the IL-23p19 gene promoter region in monocytes from patients with SLE and healthy control subjects was analyzed by chromatin immunoprecipitation (ChIP) assay. Luciferase reporter gene assays were performed to identify key drivers of IL-23p19 promoter activity. TANK-binding kinase 1 (TBK-1) protein levels were determined by Western blotting. Results ChIP assays demonstrated that IRF-3 was stably bound to the human IL-23p19 promoter in monocytes; this association increased following TLR-3 stimulation. Patients with SLE demonstrated increased levels of IRF-3 bound to the IL-23p19 promoter compared with control subjects, which correlated with enhanced IL-23p19 production in monocytes from patients with SLE. Investigations of the TLR-3-driven responses in monocytes from patients with SLE revealed that TBK-1, which is critical for regulating IRF-3 activity, was hyperactivated in both resting and TLR-3-stimulated cells. Conclusion Our results demonstrate for the first time that patients with SLE display enhanced IL-23p19 expression as a result of hyperactivation of TBK-1, resulting in increased binding of IRF-3 to the promoter. These findings provide novel insights into the molecular pathogenesis of SLE and the potential role for TLR-3 in driving this response.

Original languageEnglish
Pages (from-to)1601-1609
Number of pages9
JournalArthritis and Rheumatism
Volume64
Issue number5
DOIs
Publication statusPublished - May 2012

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Interleukin-23 Subunit p19
Interferon Regulatory Factor-3
Interleukin-23
Interleukins
Toll-Like Receptor 3
Systemic Lupus Erythematosus
Monocytes
Chromatin Immunoprecipitation
Phosphotransferases
Luciferases
Inbred C57BL Mouse
Reporter Genes
Genetic Promoter Regions
Protein Kinases
Healthy Volunteers
Western Blotting
Enzyme-Linked Immunosorbent Assay
Macrophages
Cytokines

ASJC Scopus subject areas

  • Immunology
  • Immunology and Allergy
  • Rheumatology
  • Pharmacology (medical)

Cite this

Enhanced interferon regulatory factor 3 binding to the interleukin-23p19 promoter correlates with enhanced interleukin-23 expression in systemic lupus erythematosus. / Smith, Siobhán; Gabhann, Joan Ní; Higgs, Rowan; Stacey, Kevin; Wahren-Herlenius, Marie; Espinosa, Alexander; Totaro, Maria Grazia; Sica, Antonio; Ball, Elizabeth; Bell, Aubrey; Johnston, James; Browne, Peter; O'Neill, Lorraine; Kearns, Grainne; Jefferies, Caroline A.

In: Arthritis and Rheumatism, Vol. 64, No. 5, 05.2012, p. 1601-1609.

Research output: Contribution to journalArticle

Smith, S, Gabhann, JN, Higgs, R, Stacey, K, Wahren-Herlenius, M, Espinosa, A, Totaro, MG, Sica, A, Ball, E, Bell, A, Johnston, J, Browne, P, O'Neill, L, Kearns, G & Jefferies, CA 2012, 'Enhanced interferon regulatory factor 3 binding to the interleukin-23p19 promoter correlates with enhanced interleukin-23 expression in systemic lupus erythematosus', Arthritis and Rheumatism, vol. 64, no. 5, pp. 1601-1609. https://doi.org/10.1002/art.33494
Smith, Siobhán ; Gabhann, Joan Ní ; Higgs, Rowan ; Stacey, Kevin ; Wahren-Herlenius, Marie ; Espinosa, Alexander ; Totaro, Maria Grazia ; Sica, Antonio ; Ball, Elizabeth ; Bell, Aubrey ; Johnston, James ; Browne, Peter ; O'Neill, Lorraine ; Kearns, Grainne ; Jefferies, Caroline A. / Enhanced interferon regulatory factor 3 binding to the interleukin-23p19 promoter correlates with enhanced interleukin-23 expression in systemic lupus erythematosus. In: Arthritis and Rheumatism. 2012 ; Vol. 64, No. 5. pp. 1601-1609.
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title = "Enhanced interferon regulatory factor 3 binding to the interleukin-23p19 promoter correlates with enhanced interleukin-23 expression in systemic lupus erythematosus",
abstract = "Objective To examine the role of interferon regulatory factor 3 (IRF-3) in the regulation of interleukin-23 (IL-23) production in patients with systemic lupus erythematosus (SLE). Methods Bone marrow-derived macrophages were isolated from both wild-type and IRF3 -/- C57BL/6 mice. These cells were stimulated with the Toll-like receptor 3 (TLR-3) agonist poly(I-C), and IL-23p19 cytokine levels were analyzed by enzyme-linked immunosorbent assay. IRF-3 binding to the IL-23p19 gene promoter region in monocytes from patients with SLE and healthy control subjects was analyzed by chromatin immunoprecipitation (ChIP) assay. Luciferase reporter gene assays were performed to identify key drivers of IL-23p19 promoter activity. TANK-binding kinase 1 (TBK-1) protein levels were determined by Western blotting. Results ChIP assays demonstrated that IRF-3 was stably bound to the human IL-23p19 promoter in monocytes; this association increased following TLR-3 stimulation. Patients with SLE demonstrated increased levels of IRF-3 bound to the IL-23p19 promoter compared with control subjects, which correlated with enhanced IL-23p19 production in monocytes from patients with SLE. Investigations of the TLR-3-driven responses in monocytes from patients with SLE revealed that TBK-1, which is critical for regulating IRF-3 activity, was hyperactivated in both resting and TLR-3-stimulated cells. Conclusion Our results demonstrate for the first time that patients with SLE display enhanced IL-23p19 expression as a result of hyperactivation of TBK-1, resulting in increased binding of IRF-3 to the promoter. These findings provide novel insights into the molecular pathogenesis of SLE and the potential role for TLR-3 in driving this response.",
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T1 - Enhanced interferon regulatory factor 3 binding to the interleukin-23p19 promoter correlates with enhanced interleukin-23 expression in systemic lupus erythematosus

AU - Smith, Siobhán

AU - Gabhann, Joan Ní

AU - Higgs, Rowan

AU - Stacey, Kevin

AU - Wahren-Herlenius, Marie

AU - Espinosa, Alexander

AU - Totaro, Maria Grazia

AU - Sica, Antonio

AU - Ball, Elizabeth

AU - Bell, Aubrey

AU - Johnston, James

AU - Browne, Peter

AU - O'Neill, Lorraine

AU - Kearns, Grainne

AU - Jefferies, Caroline A.

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N2 - Objective To examine the role of interferon regulatory factor 3 (IRF-3) in the regulation of interleukin-23 (IL-23) production in patients with systemic lupus erythematosus (SLE). Methods Bone marrow-derived macrophages were isolated from both wild-type and IRF3 -/- C57BL/6 mice. These cells were stimulated with the Toll-like receptor 3 (TLR-3) agonist poly(I-C), and IL-23p19 cytokine levels were analyzed by enzyme-linked immunosorbent assay. IRF-3 binding to the IL-23p19 gene promoter region in monocytes from patients with SLE and healthy control subjects was analyzed by chromatin immunoprecipitation (ChIP) assay. Luciferase reporter gene assays were performed to identify key drivers of IL-23p19 promoter activity. TANK-binding kinase 1 (TBK-1) protein levels were determined by Western blotting. Results ChIP assays demonstrated that IRF-3 was stably bound to the human IL-23p19 promoter in monocytes; this association increased following TLR-3 stimulation. Patients with SLE demonstrated increased levels of IRF-3 bound to the IL-23p19 promoter compared with control subjects, which correlated with enhanced IL-23p19 production in monocytes from patients with SLE. Investigations of the TLR-3-driven responses in monocytes from patients with SLE revealed that TBK-1, which is critical for regulating IRF-3 activity, was hyperactivated in both resting and TLR-3-stimulated cells. Conclusion Our results demonstrate for the first time that patients with SLE display enhanced IL-23p19 expression as a result of hyperactivation of TBK-1, resulting in increased binding of IRF-3 to the promoter. These findings provide novel insights into the molecular pathogenesis of SLE and the potential role for TLR-3 in driving this response.

AB - Objective To examine the role of interferon regulatory factor 3 (IRF-3) in the regulation of interleukin-23 (IL-23) production in patients with systemic lupus erythematosus (SLE). Methods Bone marrow-derived macrophages were isolated from both wild-type and IRF3 -/- C57BL/6 mice. These cells were stimulated with the Toll-like receptor 3 (TLR-3) agonist poly(I-C), and IL-23p19 cytokine levels were analyzed by enzyme-linked immunosorbent assay. IRF-3 binding to the IL-23p19 gene promoter region in monocytes from patients with SLE and healthy control subjects was analyzed by chromatin immunoprecipitation (ChIP) assay. Luciferase reporter gene assays were performed to identify key drivers of IL-23p19 promoter activity. TANK-binding kinase 1 (TBK-1) protein levels were determined by Western blotting. Results ChIP assays demonstrated that IRF-3 was stably bound to the human IL-23p19 promoter in monocytes; this association increased following TLR-3 stimulation. Patients with SLE demonstrated increased levels of IRF-3 bound to the IL-23p19 promoter compared with control subjects, which correlated with enhanced IL-23p19 production in monocytes from patients with SLE. Investigations of the TLR-3-driven responses in monocytes from patients with SLE revealed that TBK-1, which is critical for regulating IRF-3 activity, was hyperactivated in both resting and TLR-3-stimulated cells. Conclusion Our results demonstrate for the first time that patients with SLE display enhanced IL-23p19 expression as a result of hyperactivation of TBK-1, resulting in increased binding of IRF-3 to the promoter. These findings provide novel insights into the molecular pathogenesis of SLE and the potential role for TLR-3 in driving this response.

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