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A subset of CD163(+) macrophages displays mixed polarizations in discoid lupus skin.

Chong BF, Tseng LC, Hosler GA, Teske NM, Zhang S, Karp DR, Olsen NJ, Mohan C - Arthritis Res. Ther. (2015)

Bottom Line: Gene expression of RNA from DLE lesional and normal control skin was compared by microarrays and quantitative real-time polymerase chain reaction (RT-PCR).DLE skin had twice as many upregulated genes than downregulated genes compared with normal skin.Quantitative RT-PCR showed that several M1 macrophage-associated genes-e.g., chemokine (C-X-C motif) ligand 10 (CXCL10), chemokine (C-C motif) ligand 5 (CCL5), and signal transducer and activator of transcription 1 (STAT1)-had amplified mRNA levels in DLE skin.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, 75390-9069, USA. ben.chong@utsouthwestern.edu.

ABSTRACT

Introduction: Lesional skin of patients with discoid lupus erythematosus (DLE) contains macrophages, whose polarization has yet to be investigated. To test our hypothesis that M1 macrophages would be increased in DLE skin, we examined transcriptome alterations in immune cell gene expression and macrophage features in DLE and normal skin by using gene expression and histochemical approaches.

Methods: Gene expression of RNA from DLE lesional and normal control skin was compared by microarrays and quantitative real-time polymerase chain reaction (RT-PCR). Both skin groups were analyzed for CD163 expression by immunohistochemistry. Double immunofluorescence studies were performed to characterize protein expression of CD163(+) macrophages.

Results: DLE skin had twice as many upregulated genes than downregulated genes compared with normal skin. Gene set enrichment analysis comparing differentially expressed genes in DLE and normal skin with previously published gene sets associated with M1 and M2 macrophages showed strong overlap between upregulated genes in DLE skin and M1 macrophages. Quantitative RT-PCR showed that several M1 macrophage-associated genes-e.g., chemokine (C-X-C motif) ligand 10 (CXCL10), chemokine (C-C motif) ligand 5 (CCL5), and signal transducer and activator of transcription 1 (STAT1)-had amplified mRNA levels in DLE skin. CD163(+) macrophages were increased near the epidermal-dermal junction and perivascular areas in DLE skin compared with normal skin. However, double immunofluorescence studies of CD163(+) macrophages revealed minor co-expression of M1 (CXCL10, tumor necrosis factor-alpha, and CD127) and M2 (CD209 and transforming growth factor-beta) macrophage-related proteins in DLE skin.

Conclusion: Whereas a subset of CD163(+) macrophages displays mixed polarizations in DLE skin, other immune cells such as T cells can contribute to the expression of these macrophage-related genes.

No MeSH data available.


Related in: MedlinePlus

qRT-PCR analysis of selected genes in DLE lesional and normal skin. a–k RNA expression of general macrophage markers (CD163 and CD68) (a, b), M1 macrophage-related markers (CD127, TNF-α, CXCL10, STAT1, and CCL5) (c–g), and M2 macrophage-related markers (TGF-β, CD206, CD209, and arginase-1) (h–k) was compared in DLE lesional (n = 17) and normal (n = 12) skin via qRT-PCR. Multiple general and M1 macrophage-related markers were upregulated in DLE lesional skin. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. CCL chemokine (C-C motif) ligand, CXCL chemokine (C-X-C motif) ligand, DLE discoid lupus erythematosus, qRT-PCR quantitative real-time polymerase chain reaction, STAT signal transducer and activator of transcription, TGF-β transforming growth factor-beta, TNF-α tumor necrosis factor-alpha
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Fig2: qRT-PCR analysis of selected genes in DLE lesional and normal skin. a–k RNA expression of general macrophage markers (CD163 and CD68) (a, b), M1 macrophage-related markers (CD127, TNF-α, CXCL10, STAT1, and CCL5) (c–g), and M2 macrophage-related markers (TGF-β, CD206, CD209, and arginase-1) (h–k) was compared in DLE lesional (n = 17) and normal (n = 12) skin via qRT-PCR. Multiple general and M1 macrophage-related markers were upregulated in DLE lesional skin. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. CCL chemokine (C-C motif) ligand, CXCL chemokine (C-X-C motif) ligand, DLE discoid lupus erythematosus, qRT-PCR quantitative real-time polymerase chain reaction, STAT signal transducer and activator of transcription, TGF-β transforming growth factor-beta, TNF-α tumor necrosis factor-alpha

Mentions: Several macrophage-related genes were selected for confirmatory qRT-PCR studies on DLE lesional (n = 17) and normal sun-exposed (n = 12) skin. CD163 (2.83 fold change (FC)) and CD68 (4.98 FC), which are both markers used to identify macrophages, had significantly higher levels in DLE skin than normal skin (P < 0.0001) (Fig. 2a, b). Multiple M1 macrophage genes—e.g., CD127 (8.01 FC, P < 0.0001), TNF-α (1.92 FC, P = 0.047), CXCL10 (45.91 FC, P < 0.0001), STAT1 (11.96 FC, P < 0.0001), CCL5 (25.85 FC, P < 0.0001), CD86, and Mx1—were also significantly upregulated in DLE skin compared with normal skin (Fig. 2c–g and Additional file 1: Table S4). IFN-γ and IL-12, two cytokines associated with M1 macrophages, were higher in DLE skin but did not reach statistical significance (Additional file 1: Table S4). With the exception of TGF-β (2.42 FC, P = 0.0002), which was significantly higher in DLE skin than normal skin, multiple M2 macrophage genes—e.g., CD206 (0.72 FC, P = 0.32), CD209 (1.10 FC, P = 0.97), arginase-1 (1.25 FC, P = 0.19), FOLR2, and IL-10—were not differentially expressed in DLE and normal skin (Fig. 2h–k and Additional file 1: Table S4). We also selected type I interferon-inducible genes (e.g., Mx1, ISG15, and Ly6E) and genes associated with TH1 cells (e.g., CXCR3) and CD8+ T cells (e.g., CD8 and granzyme B) for qRT-PCR analysis. These were significantly elevated in DLE skin versus normal skin (P < 0.0001) (Additional file 1: Table S4).Fig. 2


A subset of CD163(+) macrophages displays mixed polarizations in discoid lupus skin.

Chong BF, Tseng LC, Hosler GA, Teske NM, Zhang S, Karp DR, Olsen NJ, Mohan C - Arthritis Res. Ther. (2015)

qRT-PCR analysis of selected genes in DLE lesional and normal skin. a–k RNA expression of general macrophage markers (CD163 and CD68) (a, b), M1 macrophage-related markers (CD127, TNF-α, CXCL10, STAT1, and CCL5) (c–g), and M2 macrophage-related markers (TGF-β, CD206, CD209, and arginase-1) (h–k) was compared in DLE lesional (n = 17) and normal (n = 12) skin via qRT-PCR. Multiple general and M1 macrophage-related markers were upregulated in DLE lesional skin. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. CCL chemokine (C-C motif) ligand, CXCL chemokine (C-X-C motif) ligand, DLE discoid lupus erythematosus, qRT-PCR quantitative real-time polymerase chain reaction, STAT signal transducer and activator of transcription, TGF-β transforming growth factor-beta, TNF-α tumor necrosis factor-alpha
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4644297&req=5

Fig2: qRT-PCR analysis of selected genes in DLE lesional and normal skin. a–k RNA expression of general macrophage markers (CD163 and CD68) (a, b), M1 macrophage-related markers (CD127, TNF-α, CXCL10, STAT1, and CCL5) (c–g), and M2 macrophage-related markers (TGF-β, CD206, CD209, and arginase-1) (h–k) was compared in DLE lesional (n = 17) and normal (n = 12) skin via qRT-PCR. Multiple general and M1 macrophage-related markers were upregulated in DLE lesional skin. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. CCL chemokine (C-C motif) ligand, CXCL chemokine (C-X-C motif) ligand, DLE discoid lupus erythematosus, qRT-PCR quantitative real-time polymerase chain reaction, STAT signal transducer and activator of transcription, TGF-β transforming growth factor-beta, TNF-α tumor necrosis factor-alpha
Mentions: Several macrophage-related genes were selected for confirmatory qRT-PCR studies on DLE lesional (n = 17) and normal sun-exposed (n = 12) skin. CD163 (2.83 fold change (FC)) and CD68 (4.98 FC), which are both markers used to identify macrophages, had significantly higher levels in DLE skin than normal skin (P < 0.0001) (Fig. 2a, b). Multiple M1 macrophage genes—e.g., CD127 (8.01 FC, P < 0.0001), TNF-α (1.92 FC, P = 0.047), CXCL10 (45.91 FC, P < 0.0001), STAT1 (11.96 FC, P < 0.0001), CCL5 (25.85 FC, P < 0.0001), CD86, and Mx1—were also significantly upregulated in DLE skin compared with normal skin (Fig. 2c–g and Additional file 1: Table S4). IFN-γ and IL-12, two cytokines associated with M1 macrophages, were higher in DLE skin but did not reach statistical significance (Additional file 1: Table S4). With the exception of TGF-β (2.42 FC, P = 0.0002), which was significantly higher in DLE skin than normal skin, multiple M2 macrophage genes—e.g., CD206 (0.72 FC, P = 0.32), CD209 (1.10 FC, P = 0.97), arginase-1 (1.25 FC, P = 0.19), FOLR2, and IL-10—were not differentially expressed in DLE and normal skin (Fig. 2h–k and Additional file 1: Table S4). We also selected type I interferon-inducible genes (e.g., Mx1, ISG15, and Ly6E) and genes associated with TH1 cells (e.g., CXCR3) and CD8+ T cells (e.g., CD8 and granzyme B) for qRT-PCR analysis. These were significantly elevated in DLE skin versus normal skin (P < 0.0001) (Additional file 1: Table S4).Fig. 2

Bottom Line: Gene expression of RNA from DLE lesional and normal control skin was compared by microarrays and quantitative real-time polymerase chain reaction (RT-PCR).DLE skin had twice as many upregulated genes than downregulated genes compared with normal skin.Quantitative RT-PCR showed that several M1 macrophage-associated genes-e.g., chemokine (C-X-C motif) ligand 10 (CXCL10), chemokine (C-C motif) ligand 5 (CCL5), and signal transducer and activator of transcription 1 (STAT1)-had amplified mRNA levels in DLE skin.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX, 75390-9069, USA. ben.chong@utsouthwestern.edu.

ABSTRACT

Introduction: Lesional skin of patients with discoid lupus erythematosus (DLE) contains macrophages, whose polarization has yet to be investigated. To test our hypothesis that M1 macrophages would be increased in DLE skin, we examined transcriptome alterations in immune cell gene expression and macrophage features in DLE and normal skin by using gene expression and histochemical approaches.

Methods: Gene expression of RNA from DLE lesional and normal control skin was compared by microarrays and quantitative real-time polymerase chain reaction (RT-PCR). Both skin groups were analyzed for CD163 expression by immunohistochemistry. Double immunofluorescence studies were performed to characterize protein expression of CD163(+) macrophages.

Results: DLE skin had twice as many upregulated genes than downregulated genes compared with normal skin. Gene set enrichment analysis comparing differentially expressed genes in DLE and normal skin with previously published gene sets associated with M1 and M2 macrophages showed strong overlap between upregulated genes in DLE skin and M1 macrophages. Quantitative RT-PCR showed that several M1 macrophage-associated genes-e.g., chemokine (C-X-C motif) ligand 10 (CXCL10), chemokine (C-C motif) ligand 5 (CCL5), and signal transducer and activator of transcription 1 (STAT1)-had amplified mRNA levels in DLE skin. CD163(+) macrophages were increased near the epidermal-dermal junction and perivascular areas in DLE skin compared with normal skin. However, double immunofluorescence studies of CD163(+) macrophages revealed minor co-expression of M1 (CXCL10, tumor necrosis factor-alpha, and CD127) and M2 (CD209 and transforming growth factor-beta) macrophage-related proteins in DLE skin.

Conclusion: Whereas a subset of CD163(+) macrophages displays mixed polarizations in DLE skin, other immune cells such as T cells can contribute to the expression of these macrophage-related genes.

No MeSH data available.


Related in: MedlinePlus