Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Silverchair Information Systems full text link Silverchair Information Systems Free PMC article
Full text links

Actions

Share

.2013 Sep 15;191(6):3139-51.
doi: 10.4049/jimmunol.1300329. Epub 2013 Aug 19.

IL-4 and retinoic acid synergistically induce regulatory dendritic cells expressing Aldh1a2

Affiliations

IL-4 and retinoic acid synergistically induce regulatory dendritic cells expressing Aldh1a2

Bing Zhu et al. J Immunol..

Abstract

Although activated inflammatory monocytes (IMCs) and inflammatory dendritic cells (IDCs) are potent T cell suppressors, nonactivated IMCs and IDCs promote T cell activation and Th1/Th17 cell differentiation. In this study, we investigated how to reduce the proinflammatory properties of IMCs and IDCs and further convert them into immune regulatory dendritic cells (DCs). We found that IL-4 and retinoic acid (RA) cotreatment of GM-CSF-differentiated IDCs synergistically induced the expression of aldehyde dehydrogenase family 1, subfamily A2, a rate-limiting enzyme for RA synthesis in DCs. IL-4 plus RA-treated IDCs upregulated CD103 expression and markedly reduced the production of proinflammatory cytokines upon activation. IL-4 plus RA-treated IDCs strongly induced CD4⁺Foxp3⁺ regulatory T cell differentiation and suppressed Th1 and Th17 differentiation. Mechanistically, the transcription factors Stat6 and RA receptor β play important roles in aldehyde dehydrogenase family 1, subfamily A2, induction. In addition, IL-4 and RA signaling pathways interact closely to enhance the regulatory function of treated DCs. Adoptive transfer of IL-4 plus RA-treated DCs significantly increased regulatory T cell frequency in vivo. Direct treatment with IL-4 and RA also markedly suppressed actively induced experimental autoimmune encephalomyelitis. Our data demonstrate the synergistic effect of IL-4 and RA in inducing a regulatory phenotype in IDCs, providing a potential treatment strategy for autoimmune diseases.

PubMed Disclaimer

Figures

Figure 1
Figure 1
IL-4 and RA co-treatment synergistically induces Aldh1a2 expression in IDCs. A) IDCs were treated with IL-4 (20 ng/ml) and/or RA (1 μM) for 24 h, andAldh1a2 mRNA was quantified by RT-PCR. B) Aldh1a2 protein expression was examined by immunoblotting in non-treated and IL-4 and/or RA-treated IDCs at 24 h. C) Aldehyde dehydrogenase activity was examined by ALDEFLUOR staining in IDCs treated for 24 h. Data quantifying the median fluorescence intensity are shown in the right graph. D) IDCs were treated with IL-4 and RA for 24 h. After washing, cells were either non-activated or activated with IFN-γ (20 ng/ml) and agonistic anti-CD40 (20 μg/ml) for 24 h.Aldh1a2 expression was quantified by RT-PCR. E) Culture supernatants collected from experiments described in D) were analyzed by Milliplex assay for cytokine production. F) Phenotype of IDCs was analyzed by flow cytometry after treatment for 24 h. Expression of CD11c and CD103 is shown in solid lines, and isotype control staining is shown in tinted gray areas. *, P < 0.05; #, P < 0.01; n.s., not significant. Labels over individual columns indicate the statistical significance compared to the first column on left (non-treated group). Labels on the connecting lines show the statistical significance among other columns. Data are representative of two to three independent experiments.
Figure 2
Figure 2
IL-4+RA-treated IDCs inhibit T cell production of IFN-γ and IL-17. A) CD4+ T cells from 2D2 MOG TCR transgenic mice were cultured with non-treated IDCs or IDCs treated with IL-4, RA, or IL-4+RA in the presence of MOG35-55 peptide (20 μg/ml). T cell proliferation assay was performed after 48 h. B) IL-2, IFN-γ and IL-17 concentrations in the culture supernatants of above experiments were measured. C) Naïve 2D2 CD4+ T cells were cultured with various IDCs in neutral (Th0) or Th17 polarizing condition for 72 h. Intracellular staining for IFN-γ and IL-17 was examined in gated CD4+ T cells. D) Intracellular staining of 2D2 CD4+ T cells after cultured with control IDCs and/or IL-4+RA-treated IDCs in Th17 polarizing condition. LE135 (RAR antagonist), L-NIL (NOS2 inhibitor) and nor-NOHA (Arginase I inhibitor) were used at 1 μM, 1 mM, and 0.5 mM respectively. In these experiments, T cells were used at 1 × 105/well, and IDCs were used at 5 × 104/well in a round bottom 96-well plate. *, P < 0.05; n.s., not significant. Data are representative of three independent experiments.
Figure 3
Figure 3
IL-4+RA-treated IDCs promote Treg differentiation through RA production. A) CD4+ T cells from 2D2 Foxp3 KI mice were cultured with various IDCs in Treg polarizing condition for 72 h. Foxp3+GFP+ cell frequency was examined in gated CD4+ T cells by flow cytometry. B) Frequencies of Foxp3+GFP+ and IL-17-producing CD4+ T cells were determined after co-culturing with control or IL-4+RA-treated IDCs in Th17 polarizing condition. C) CD4+Foxp3−cells were isolated from 2D2 Foxp3 KI mice and cultured with various IDCs under Treg polarizing condition. Efficiency of conversion from Foxp3- to Foxp3+ T cells was examined. D) Naturally occurring CD4+Foxp3+ Treg cells were isolated from 2D2 Foxp3 KI mice, and cultured with control or IL-4+RA-treated IDCs under Treg polarizing condition. Proliferation assay was performed after 48 h. In these experiments, T cells were used at 1 × 105/well, and IDCs were used at 5 × 104/well in a round bottom 96-well plate. #, P < 0.01. Data are representative of three independent experiments.
Figure 4
Figure 4
Role of Stat6 activation in the induction of Aldh1a2 expression. A) IDC protein samples were collected after IL-4+RA treatment for 0 to 2 h. Stat6 phosphorylated at Tyr641 and total Stat6 was quantified by immunoblotting. B) IDCs from wild type orStat6−/− mice were treated with IL-4, RA or IL-4+RA for 24 h.Aldh1a2 expression in treated IDCs was compared to non-treated IDCs by RT-PCR. C) BMDCs derived from wild-type B6 mice were treated with IL-4+RA for 3h. ChIP analysis was performed with control IgG or anti-Stat6 antibody, and PCR was performed with primers specific for two sites within 1-kb upstream ofAldh1a2. D) ChIP PCR was performed as above, with BMDCs derived fromStat6−/− mice, and. #, P < 0.01; n.s., not significant. Data are representative of two independent experiments.
Figure 5
Figure 5
Expression and role of RARβ in IL-4+RA-treated IDCs. A) Expression ofRara,Rarb andRarg in control IDCs and those treated with IL4, RA or IL-4+RA for 24 h was examined by RT-PCR. B) Time course ofRarb expression in IL-4+RA-treated IDCs was determined by RT-PCR. C) Expression of RARβ was measured in nuclear protein extracts from control and treated IDCs at 24 h by immunoblotting. D) BMDCs were treated with IL-4+RA for 3 h. ChIP PCR was performed with control IgG or anti-RARβ antibody, and PCR primers were specific for two sites within 1-kb upstream ofRarb. E) ChIP experiment was the same as in D), with PCR primers specific for two sites within 1-kb upstream ofAldh1a2. *, P < 0.05; #, P < 0.01; n.s., not significant. Data are representative of two to three independent experiments.
Figure 6
Figure 6
Interaction of IL-4 and RA signaling pathways. A) IDCs were derived from wild type andStat6−/− mice, and treated with IL-4, RA, or IL-4+RA for 24 h.Rarb expression was compared with non-treated IDCs by RT-PCR. B) BMDCs from wild type mice were treated with IL-4+RA for 3 h. ChIP PCR was performed with control IgG and anti-Stat6 antibody, and PCR primers were specific for the two sites within 1-kb upstream ofRarb. C) ChIP experiment was the same as in B), with BMDCs derived fromStat6−/− mice. D) IDCs from wild type mice were treated with RA for 24 h.Il4ra expression was compared to non-treated IDCs by RT-PCR. E) BMDCs from wild type mice were treated with IL-4+RA for 3 h. ChIP PCR was performed with control IgG and anti-RARβ antibody, and PCR primers were specific for two sites within 1-kb upstream ofIl4ra. *, P < 0.05; #, P < 0.01; n.s., not significant. Data are representative of two to three independent experiments.
Figure 7
Figure 7
Role of Stat6 and RARβ in activatingAldh1a2 promoter. A) RAW264.7 cells were transfected withStat6 and/orRarb expression plasmids,Aldh1a2 promoter reporter plasmid and Renilla luciferase plasmid.Aldh1a2 promoter activity was examined by luciferase assay after 48 h. B) The same luciferase assay was performed as above, and RAW264.7 cells were treated with IL-4+RA after plasmid transfection. #, P < 0.01; n.s., not significant. Data are representative of three independent experiments.
Figure 8
Figure 8
Transfer of IL-4+RA-treated BMDCs increases Treg frequencyin vivo. A) Control IDCs and IL-4+RA-treated IDCs were pulsed with MOG35-55 peptide (20 μg/ml) for 4 h and transferred into naïve 2D2 mice at 107 cell/mouse on days 0 and 4. On day 7, splenocytes and blood leukocytes were harvested, and Foxp3+ cell frequency in CD4+ T cells was determined by flow cytometry. Quantification data are shown in graphs on the right. B) IFN-γ and IL-17 production from splenic CD4+ T cells was examined by intracellular cytokine staining and flow cytometry. Quantification data are shown in the graph on the right. *, P < 0.05. Data are representative of two independent experiments with 3 mice per group in each experiment.
Figure 9
Figure 9
Co-treatment with IL-4 and RA effectively suppresses EAE. A) B6 mice were immunized with MOG35-55 on day 0 to induce EAE, and received daily treatment from day 0 to day 15. Five groups were set up: non-treated, vehicle-treated, IL-4-treated, RA-treated and IL-4+RA-treated. Data were pooled from 2 independent experiments showing similar results. B) Spinal cord tissues were harvested on day 20 from 3 mice per group, and processed for H&E and Luxol fast blue staining. The number of inflammatory foci and the percentage of white matter area with demyelination were quantified. C) Splenocytes were harvested on day 20 from 3 mice per group, and activated with 20 μg/ml MOG35-55 for 48 hin vitro. Cytokine production in the supernatant was examined with Milliplex assays. D) Splenic CD11b+Ly-6Chi cells were isolated on day 20, and mRNAs ofAldh1a2 andRarb were quantified by RT-PCR. *, P < 0.05; #, P < 0.01.
Figure 10
Figure 10
Illustration of IL-4 and RA signaling pathways leading to Aldh1a2 induction. Increased RA production from IDCs promotes Treg differentiation and suppresses Th1 and Th17 differentiation.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Geissmann F, Auffray C, Palframan R, Wirrig C, Ciocca A, Campisi L, Narni-Mancinelli E, Lauvau G. Blood monocytes: distinct subsets, how they relate to dendritic cells, and their possible roles in the regulation of T-cell responses. Immunol Cell Biol. 2008;86:398–408. - PubMed
    1. Shi C, Pamer EG. Monocyte recruitment during infection and inflammation. Nat Rev Immunol. 2011;11:762–774. - PMC - PubMed
    1. Sunderkotter C, Nikolic T, Dillon MJ, Van Rooijen N, Stehling M, Drevets DA, Leenen PJ. Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response. J Immunol. 2004;172:4410–4417. - PubMed
    1. Zhu B, Bando Y, Xiao S, Yang K, Anderson AC, Kuchroo VK, Khoury SJ. CD11b+Ly-6C(hi) suppressive monocytes in experimental autoimmune encephalomyelitis. J Immunol. 2007;179:5228–5237. - PubMed
    1. Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity. 2003;19:71–82. - PubMed

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full text links
Silverchair Information Systems full text link Silverchair Information Systems Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp