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, 6 (2), e1181237
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IL-32 Induces Indoleamine 2,3-dioxygenase + CD1c + Dendritic Cells and Indoleamine 2,3-dioxygenase + CD163 + Macrophages: Relevance to Mycosis Fungoides Progression

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IL-32 Induces Indoleamine 2,3-dioxygenase + CD1c + Dendritic Cells and Indoleamine 2,3-dioxygenase + CD163 + Macrophages: Relevance to Mycosis Fungoides Progression

Hanako Ohmatsu et al. Oncoimmunology.

Abstract

Mycosis fungoides (MF) progresses from patch to tumor stage by expansion of malignant T-cells that fail to be controlled by protective immune mechanisms. In this study, we focused on IL-32, a cytokine, highly expressed in MF lesions. Depending on the other cytokines (IL-4, GM-CSF) present during in vitro culture of healthy volunteers' monocytes, IL-32 increased the maturation of CD11c+ myeloid dendritic cells (mDC) and/or CD163+ macrophages, but IL-32 alone showed a clear ability to promote dendritic cell (DC) differentiation from monocytes. DCs matured by IL-32 had the phenotype of skin-resident DCs (CD1c+), but more importantly, also had high expression of indoleamine 2,3-dioxygenase. The presence of DCs with these markers was demonstrated in MF skin lesions. At a molecular level, indoleamine 2,3-dioxygenase messenger RNA (mRNA) levels in MF lesions were higher than those in healthy volunteers, and there was a high correlation between indoleamine 2,3-dioxygenase and IL-32 expression. In contrast, Foxp3 mRNA levels decreased from patch to tumor stage. Increasing expression of IL-10 across MF lesions was highly correlated with IL-32 and indoleamine 2,3-dioxygenase, but not with Foxp3 expression. Thus, IL-32 could contribute to progressive immune dysregulation in MF by directly fostering development of immunosuppressive mDC or macrophages, possibly in association with IL-10.

Keywords: CD163+ macrophages; CD1c+ dendritic cells; IL-32; indoleamine 2,3-dioxygenase (IDO); mycosis fungoides.

Figures

Figure 1.
Figure 1.
CD11c+ as well as CD1c+ cells in MF skin. (A) Immunohistochemistry for CD11c using skin from mycosis fungoides (MF) patch stage, MF plaque stage, and MF tumor stage. (B) Immunohistochemistry for CD1c using skin from MF patch stage, MF plaque stage, and MF tumor stage. (A and B) Magnification, x10. Scale bars represent 100 μm.
Figure 2.
Figure 2.
Changes of CD14, CD163, and CD68 expression on monocyte-derived cells cultured with the addition of IL-32 β or IL-32 γ. Flow cytometry analysis of the monocyte and macrophage markers (A) CD14, (B) CD163, and (C) CD68 on human monocytes stimulated for 5 d with the control medium with nothing (nothing), 25 ng/mL IL-4 (IL-4), 100 IU/mL GM-CSF (GM-CSF), or 25 ng/mL IL-4 + 100 IU/mL GM-CSF (IL-4 + GM-CSF) with or without 50 ng/mL IL-32β (left panels) or IL-32γ (right panels). Gray and black lines represent the results of cells obtained from the control medium without IL-32 and the control medium with IL-32β or IL-32γ, respectively. (A) Viable (LIVE/DEAD) cells among all harvested cells were selected to show CD14 expression. (B and C) Viable CD14+ cells were selected to be analyzed. One Fluorescence minus one (FMO) is shown to represent the specificity of the antibodies as a dot line.
Figure 3.
Figure 3.
Changes of CD86 and CD83 expression on CD14HLA-DRhighCD11c+ cells by addition of IL-32. Cytokines added to the control medium are written to the left of the panels. (A) Viable CD14 cells harvested from the control medium with nothing or from the control media with the addition of IL-32 were selected to detect HLA-DR+CD11c+ cells. The left panels show the cells obtained from the control medium. The middle and right panels represent the cells obtained from the control medium with IL-32β or IL-32γ, respectively. The numbers written within the panels represent the percentages of the circled population (HLA-DR+CD11c+ cells). (B) CD86 and CD83 expression on viable CD14HLA-DRhighCD11c+ cells (coming from the circled gate in the corresponding condition of (A)). Red and blue lines represent the cells harvested from the control medium without IL-32 and the control medium with IL-32β or IL-32γ, respectively. One FMO is shown to represent the specificity of CD83 antibody as a dot line.
Figure 4.
Figure 4.
Increase in CD1c+ cells from monocytes cultured with the addition of IL-32. Cytokines added to the control medium are shown to the left of the panels. (A) Expression of CD1c on viable CD14HLA-DR+CD11c+ cells. Red and blue lines represent the cells harvested from the control medium without IL-32 and with IL-32β or IL-32γ, respectively. (B) Viable CD14HLA-DRhigh cells were selected to show CD11c (x-axis) and CD1c (y-axis) expression. The left panels show the cells obtained from the control medium. The middle and the right panels represent the cells obtained from the control medium with IL-32β or IL-32β, respectively. The numbers within the panels show the percentages of the circled population (CD11c+CD1c+ cells).
Figure 5.
Figure 5.
IDO expression in MF skin. (A) IDO mRNA expression using skin samples (VL; healthy volunteers, MF; mycosis fungoides/patch; patch stage, plaque; plaque stage, tumor; tumor stage). Values are mean ± SD. ***p <0.001. (B) Correlations between mRNA expression levels of IDO (y-axis) and IL-32 (x-axis) in MF skin. *p <0.05. (C) Immunohistochemistry for IDO using skin from healthy volunteer and MF patient. Magnification, x10. Scale bars represent 100 μm. (D) Alexa flour 488 (A-488)-labeled IDO (left panels) and Alexa flour 568 (A-568)-labeled CD11c, CD1c, or CD163 (center panels), with merged images shown in right panels. Magnification, x20. Scale bars represent 100 μm.
Figure 6.
Figure 6.
Increase in IDO expression on mDCs and macrophages by addition of IL-32. IDO expression on (A) Viable CD14HLA-DRhighCD11c+ cells and (B) Viable CD14+CD163+CD68+ cells. Red and blue lines represent the cells harvested from the control medium without IL-32 and with IL-32β or IL-32γ, respectively. Cytokines added to the control medium are written to the left of the panels.
Figure 7.
Figure 7.
Foxp3 and IL-10 expression in MF skin. (A) Foxp3 mRNA expression levels and the correlation between Foxp3 (y-axis) and IDO (x-axis) mRNA expression using skin samples (VL; healthy volunteers, MF; mycosis fungoides/patch; patch stage, plaque; plaque stage, tumor; tumor stage). Values are mean ± SD. *p < 0.05, ***p <0.001. (B) IL-10 mRNA expression levels using skin samples and the correlation between mRNA expression levels of IL-10 (y-axis) and IDO (x-axis). ***p < 0.001. (C) Correlations between mRNA expression levels of IL-10 (y-axis) and IL-32 or Foxp3 (x-axis). ***p < 0.001.

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Grant support

This study was partially supported by the TTCL (CTSA, RUCCTS Grant no.8 UL1 TR000043) from the National Center for Advancing Translational Sciences (NCATS, NIH). N.G. was supported by NIH MSTP grant no. GM07739.
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