Yonsei Med J.  2016 Nov;57(6):1435-1445. 10.3349/ymj.2016.57.6.1435.

Upregulation of CD47 in Regulatory T Cells in Atopic Dermatitis

• Lee N
• Shin JU
• Jin S
• Yun KN
• Kim JY
• Park CO
• Kim SH
• Noh JY
• Lee KH

• KMID: 2427162
• DOI: http://doi.org/10.3349/ymj.2016.57.6.1435

Abstract

PURPOSE
Regulatory T (Treg) cells are key modulators in the immune system. Recent studies have shown that atopic dermatitis (AD) patients have higher numbers of Treg cells; however, little is known about the specific phenotype and function of Treg cells in AD.
MATERIALS AND METHODS
To identify differentially expressed proteins in peripheral induced Treg cells in AD and naturally derived Treg cells in normal controls, CD4⁺CD25⁺ Treg cells were isolated from thymus tissue of normal mice and the spleens of AD mice. Membrane proteins were extracted, and quantitative proteomics labeling with Tandem Mass Tags (TMT) was performed, followed by one-dimensional liquid chromatography/tandem mass spectrometry analysis.
RESULTS
Using TMT labeling, we identified 510 proteins, including 63 membrane proteins and 16 plasma membrane proteins. CD47 was one of the upregulated proteins in Treg cells in AD spleens. Although CD47 was expressed in all CD4⁺ and CD8⁺ T cells, a significantly higher expression of CD47 was observed in the Treg cells of AD mice and AD patients than in those of normal mice and healthy controls. Furthermore, Treg cells from the spleen showed a significantly higher expression of CD47 than those from the thymus.
CONCLUSION
We found that CD47 is highly expressed in the Treg cells of AD mice, particularly in the spleen. Based on our results, we propose that CD47(high) Treg cells are likely induced Treg cells and that upregulated CD47 in the Treg cells of AD patients may play a role in the increased population of Treg cells in AD.

Keyword

Atopic dermatitis; regulatory T cells; CD47; Tandem Mass Tags (TMT)

MeSH Terms

Animals
CD4 Antigens/analysis
CD47 Antigen/*metabolism
Dermatitis, Atopic/immunology
Female
Humans
Mice
Severity of Illness Index
Spleen/cytology/immunology
T-Lymphocytes/drug effects/*immunology
T-Lymphocytes, Regulatory/cytology/immunology/*metabolism
*Up-Regulation
CD4 Antigens
CD47 Antigen

Figure

• 

  Fig. 1 Induction of AD-like skin lesions in NC/Nga mice and isolation of CD4+CD25+ Treg cells using MACS®. (A) Dorsal skin of mice treated with D. farinae extracts for 8 weeks. Significant erythema and crusts were observed after 3 weeks of D. farinae application. After 6 weeks of application, the most severe erythema, crusts, excoriation, and oozing were apparent. (B) SCORAD scores were plotted against the time of repeated topical application of D. farinae. (C) Serum IgE levels were measured via ELISA after repeated topical application of D. farinae ointment. All results are representative or mean±SD from groups that contained five mice. (D) CD4+CD25+ Treg cells were sorted using an AutoMACS cell sorter. The purity of isolated cells was 79.5%, and 94.7% of the isolated CD4+CD25+ Treg cells expressed Foxp3. Results are representative of three independent experiments. AD, atopic dermatitis; Treg, regulatory T; D. farinae, Dermatophagoides farinae; IgE, immunoglobulin E; ELISA, enzyme-linked immunosorbent assay.

• 

  Fig. 2 Expression of CD47 in various T cell subtypes. (A) FACS analysis of expression of CD47 in T cells, CD4+ T cells, and CD8+ T cells. All CD4+ T cells and CD8+ T cells expressed CD47. (B) FACS analysis of CD47 expression in CD3+CD4+CD25+ T cells and CD3+CD4+CD25+Foxp3+ T cells. Most CD4+CD25+Foxp3+ Treg cells expressed CD47. Treg, regulatory T; AD, atopic dermatitis.

• 

  Fig. 3 Increased expression of CD47 in Treg cells from AD mice. (A) FACS analysis of expression of CD47 in Treg cells from thymuses and spleens from normal and AD mice. The ΔMFI of CD47 was determined in Treg cells. The green line indicates CD47 in a normal thymus, the orange line indicates CD47 in a normal spleen, the blue line indicates CD47 in an AD thymus, and the red line indicates CD47 in an AD spleen. (B) CD47 expression was more upregulated in Treg cells isolated from normal spleens than in those isolated from normal thymuses. Moreover, its expression was further upregulated in Treg cells from AD spleens. (C) Western blot analysis of the expression of CD47 on Treg cells from spleens and thymuses of AD mice and normal mice. The expression level of CD47 was similar to the results of the FACS analysis. (D) Western blot densitometry. Differences were determined via one-way ANOVA. For each group, mice n=4. *p<0.05, †p<0.01, ‡p<0.001. AD, atopic dermatitis; Treg, regulatory T.

• 

  Fig. 4 Increased expression of CD47 in human Treg cells from AD patients. (A) All CD4+CD25+Foxp3+ Treg cells expressed CD47 in humans, a result similar to that for mice. (B) The expression level of CD47 was higher in Treg cells from AD patients than in those from healthy controls. (C) The severity of AD correlated with the expression of CD47 (Pearson correlation r=0.84, p=0.018). AD, atopic dermatitis; Treg, regulatory T.

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