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Induction of PIR-A/B+ DCs in the in vitro inflammatory condition and their immunoregulatory function

  • Original Article—Alimentary Tract
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Abstract

Background

Dendritic cells (DCs), primary antigen-presenting cells, are now well known as an immunoregulator of many aspects of immune responses including inflammatory bowel diseases (IBDs) such as Crohn’s disease and ulcerative colitis. We have reported that PIR-A/Bhigh cDCs (conventional DCs) appeared in dextran sodium sulfate (DSS)-induced colitis and serve as a negative immunoregulator in an animal model of IBD. The immunoregulatory role of PIR-A/B+ cDCs was confirmed in both an in vitro culture system and an in vivo transfer experiment. Here, we have investigated the differentiation process of PIR-A/B+ cDCs in an in vitro inflammatory environment and examined their functions.

Methods

cDCs were isolated from the large intestinal lamina propria from C57BL/6 mice and cultured in an inflammatory environment (IL-1, IL-6, TNFα, and LPS). The appearance of PIR-A/B+ cDCs was determined after 24 h, and the in vitro-induced PIR-A/B+ cDCs were functionally and genetically examined.

Results

PIR-A/B+ cDCs were detected after a 24-h culture only in the inflammatory environment, and the cells acted as a negative immunoregulator when examined in an allogenic mixed leukocyte reaction (MLR). The message level of IL-27 was highly upregulated in PIR-A/B+ cDCs, while that of high mobility group box 1 protein (HMGB1) was downregulated in these cells. This was well in accordance with the fact that PIR-A/B+ cDCs showed a suppressive function against activated T cells. We found that PIR-A/B+ cDCs produced IL-27, as verified by an ELISA assay, and that the inhibitory effect by PIR-A/B+ cDCs was, at least partially, due to IL-27. Furthermore, CD85d+ cells, a human counterpart of mouse PIR-A/B+ cDCs, were found in the lamina propria of the colon of the patients with ulcerative colitis, but not in the similar part of the non-inflammatory area of colon specimens from patients with colon cancer.

Conclusions

PIR-A/B+ cDCs induced in an in vitro inflammatory environment model showed a suppressive function against activated T cells by producing an inhibitory cytokine.

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References

  1. Steinman RM, Hawiger D, Liu K, et al. Dendritic cell function in vivo during the steady state: a role in peripheral tolerance. Ann N Y Acad Sci. 2003;987:15–25.

    Article  CAS  Google Scholar 

  2. Steinman RM, Hemmi H. Dendritic cells: translating innate to adaptive immunity. Curr Top Microbiol Immunol. 2006;311:17–56.

    CAS  PubMed  Google Scholar 

  3. Kretschmer K, Apostlou I, Hawiger D, et al. Inducing and expanding regulatory T cell population by foreign antigen. Nat Immunol. 2005;6:1219–27.

    Article  CAS  Google Scholar 

  4. Steinman RM, Turley S, Mellman I, et al. The induction of tolerance by dendritic cells that have captured apoptotic cells. J Exp Med. 2000;191:411–6.

    Article  CAS  Google Scholar 

  5. Molleri AE, Thomson AW. Dendritic cells: regulators of alloimmunity and opportunities for tolerance induction. Immunol Rev. 2003;196:125–46.

    Article  Google Scholar 

  6. Wakkach A, Fournier N, Brun V, et al. Characterization of dendritic cells that induce tolerance and T regulatory 1 cell differentiation in vivo. Immunity. 2003;18:605–17.

    Article  CAS  Google Scholar 

  7. Chorny A, Gonzalez-Rey E, Fernandez-Martin A, et al. Vasoactive intestinal peptide induce regulatory dendritic cells that prevent acute graft-versus-host disease while maintaining the graft-versus-tumor response. Blood. 2006;107:3787–94.

    Article  CAS  Google Scholar 

  8. Sato K, Yamashita N, Baba M, et al. Regulatory dendritic cells protect mice from murine acute graft-versus-host disease and leukemia relapse. Immunity. 2003;18:367–79.

    Article  CAS  Google Scholar 

  9. Sato K, Yamashita N, Baba M, et al. Modified myeloid dendritic cells act as regulatory dendritic cells to induce anergic and regulatory T cells. Blood. 2003;101:3581–9.

    Article  CAS  Google Scholar 

  10. Fujita S, Sato Y, Sato K, et al. Regulatory dendritic cells protect against cutaneous chronic graft-versus-host disease mediated through CD4+CD25+Foxp3+ regulatory T cells. Blood. 2007;110:3793–803.

    Article  CAS  Google Scholar 

  11. Hoshino S, Inaba M, Iwai H, et al. The role of dendritic cell subsets in 2,4,6-trinitrobenzene sulfonic acid-induced ileitis. J Autoimmun. 2010;34:380–9.

    Article  CAS  Google Scholar 

  12. Hoshino S, Kurishima A, Inaba M, et al. Amelioration of 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice by immunoregulatory dendritic cells. J Gastroenterol. 2011;46:1368–81.

    Article  CAS  Google Scholar 

  13. Kurishima A, Inaba M, Sakaguchi Y, et al. Immunoregulatory function of PIR-A/B+ DCs in the inflammatory response of dextran sodium sulfate-induced colitis. J Gastoenterol. 2014;49:1367–77.

    Article  CAS  Google Scholar 

  14. Berndt BE, Zhang M, Chen GH, et al. The role of dendritic cells in the development of acute dextran sulfate sodium colitis. J Immunol. 2007;179:6255–62.

    Article  CAS  Google Scholar 

  15. Danese S. Nonimmune cells in inflammatory bowel disease: from victim to villain. Trends Immunol. 2008;29:555–64.

    Article  CAS  Google Scholar 

  16. Rescigno M, Di Sabatino A. Dendritic cells in intestinal homeostasis and disease. J Clin Invest. 2009;119:2441–50.

    Article  CAS  Google Scholar 

  17. Abe K, Nguyen KP, Fine SD, et al. Conventional dendritic cells regulate the outcome of colonic inflammation independently of T cells. Proc Natl Acad Sci USA. 2007;104:17022–7.

    Article  CAS  Google Scholar 

  18. Fahlén L, Read S, Gorelik L, et al. T cells that cannot respond to TGF-β escape control by CD4+ CD25+ regulatory T cells. J Exp Med. 2005;201:737–46.

    Article  Google Scholar 

  19. Suri-Payer E, Cantor H. Differential cytokine requirements for regulation of autoimmune gastritis and colitis by CD4+ CD25+ T cells. J Autoimmun. 2001;16:115–23.

    Article  CAS  Google Scholar 

  20. Groux H, O’Garra A, Bigler M, et al. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature. 1997;389:737–42.

    Article  CAS  Google Scholar 

  21. Bilsborough J, George TC, Norment A, et al. Mucosal CD8alpha+ DC, with a plasmacytoid phenotype, induce differentiation and support function of T cells with regulatory properties. Immunology. 2003;108:481–92.

    Article  CAS  Google Scholar 

  22. Matteoli G, Mazzini E, Iliev ID, et al. Gut CD103+ dendritic cells express indoleamine 2,3-dioxygenase which influences T regulatory/T effector cell balance and oral tolerance induction. Gut. 2010;59:595–604.

    Article  CAS  Google Scholar 

  23. Villarino AV, Hunter CA. Biology of recently discovered cytokines: discerning the pro- and anti-inflammatory properties of interleukin-27. Arthritis Res Ther. 2004;6:225–33.

    Article  CAS  Google Scholar 

  24. Chen Y, Jiang G, Yang H-R, et al. Distinct response of liver myeloid dendritic cells to endotoxin is mediated by IL-27. J Hepatol. 2009;51:510–9.

    Article  CAS  Google Scholar 

  25. Dann SM, Le C, Choudhury BK, et al. Attenuation of intestinal inflammation in interleukin-10-deficient mice infected with Citrobacter rodentium. Infect Immun. 2014;81:1949–58.

    Article  Google Scholar 

  26. Mora JR, Bono MR, Manjunath N, Weninger W, et al. Selective imprinting of gut-homing T cells by Peyer’s patch dendritic cells. Nature. 2003;424:88–93.

    Article  CAS  Google Scholar 

  27. Laffont S, Siddiqui KR, Powrie F. Intestinal inflammation abrogates the tolerogenic properties of MLN CD103+ dendritic cells. Eur J Immunol. 2010;40:1877–83.

    Article  CAS  Google Scholar 

  28. Siddiqui KR, Laffont S, Poerie F. E-cadherin marks a subset of inflammatory dendritic cells that promote T cell-mediated colitis. Immunity. 2010;32:557–67.

    Article  CAS  Google Scholar 

  29. Mascarell L, Airouche S, Berjont N, et al. The regulatory dendritic cell marker C1q is a potent inhibitor of allergic inflammation. Mucosal Immunol. 2017;10(3):695–704. https://doi.org/10.1038/mi.2016.87 (Epub 2016 Oct 12).

    Article  CAS  PubMed  Google Scholar 

  30. Uto T, Fukaya T, Takagi H, et al. Clec4A4 is a regulatory receptor for dendritic cells that impairs inflammation and T-cell immunity. Nat Commun. 2016;12(7):11273. https://doi.org/10.1038/ncomms11273.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank Dr. Hiroyuki Gonda, Central Research of Laboratory, for his help with the flow cytometry analyses. We also thank Mari Mino-Kendson MD., Dept. Pathology, Massachusetts General Hospital, for her helpful advice in pathological studies.

Author information

Authors and Affiliations

  1. Division of Gastroenterology and Hepatology, The Third Department of Internal Medicine, Kansai Medical University, 2-5-1, Shinmachi, Hirakata, Osaka, 573-1191, Japan

    Fumi Matsui, Kazushige Uchida, Akiyoshi Nishio, Toshiro Fukui & Kazuichi Okazaki

  2. First Department of Internal Medicine, Kansai Medical University, 2-5-1, Shinmachi, Hirakata, Osaka, Japan

    Muneo Inaba, Hideaki Yoshimura, Atsushi Satake & Shosaku Nomura

  3. Gastrointestinal Surgery, Kansai Medical University, 2-5-1, Shinmachi, Hirakata, Osaka, Japan

    Kazuhiko Yoshioka

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  1. Fumi Matsui
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Correspondence to Kazuichi Okazaki.

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Electronic supplementary material

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535_2018_1447_MOESM1_ESM.jpg

Supplementary Figure 1. Appearance of PIR-A/B+ cDC subset in mice with DSS-induced colitis. The colon-derived DC-enriched population was stained with FITC–anti-CD11c, PE–anti-PIR-A/B, and APC–anti-CD45R mAbs, and the cells with the PIR-A/B+ immunophenotypes in the B220- (CD11c+) DCs were determined on days 0 (before the induction of colitis, A) and 5 after the treatment of DSS (B). The result shown here is representative of three replicate experiments. Dead cells were gated out by stain with propidium iodide. (JPEG 333 kb)

535_2018_1447_MOESM2_ESM.jpg

Supplementary Figure 2. Effect of IFNα in the induction of PIR-A/B+ cDCs. The colon-derived cDCs were prepared from normal mice, and PIR-A/B- cDCs were sorted using a FACSAria. The sorted PIR-A/B- cDCs were cultured with [GM-CSF (1μg/m/l) + IL-1 (10 ng/ml) + IL-6 (10 ng/ml) + TNFα (10 ng/ml)] (A), [GM-CSF + IL-1+ IL-6 + TNFα + IFNα (10 ng/ml)] (B), or [GM-CSF + IL-1 + IL-6 + TNFα + LPS (100 ng/ml)] (C). The DCs were harvested 48 hours later and the frequency of PIR-A/B+ cDCs (shown as gate R3 in the dot plot) were assessed using a FACS Calibur HG™ after staining with DCs with FITC–anti-CD11c mAb and PE–anti-PIR-A/B mAb. (JPEG 368 kb)

535_2018_1447_MOESM3_ESM.jpg

Supplementary Figure 3. Heat map and scatter plot. A heat map of cytokine-related genes is shown in A, and a scatter plot of PIR-A/B+ cDCs versus PIR-A/B- cDCs is shown in B. (JPEG 354 kb)

535_2018_1447_MOESM4_ESM.jpg

Supplementary Figure 3. Heat map and scatter plot. A heat map of cytokine-related genes is shown in A, and a scatter plot of PIR-A/B+ cDCs versus PIR-A/B- cDCs is shown in B. (JPEG 654 kb)

535_2018_1447_MOESM5_ESM.jpg

Supplementary Figure 4. Assessment of the inhibitory function of purified PIR-A/B+ cDCs by CFSE assay. The inhibitory function of purified PIR-A/B+ cDCs was examined by CFSE assay. Purified CD4+ T cells were stained with a CFSE reagent before the MLR culture. Four days later, the proliferative activity of CD4+ T cells was assessed using a FACSCanto II. In the histogram, the region M1 shows highly proliferative cells, and M2 shows moderately proliferative cells. The number in the figure shows the percentage of cells in each region (means and standard deviations of triplicated cultures. $, **, ##: p<0.01, #; p<0.05). It is noted that the stimulatory activity of PIR-A/B+ cDCs (E) is lower than that of splenic DCs (B). (JPEG 513 kb)

535_2018_1447_MOESM6_ESM.jpg

Supplementary Figure 5. Effect of anti-TGF-β Ab on the inhibitory function of PIR-A/B+ cDCs. Effect of anti-TGF-β Ab on the inhibitory function of PIR-A/B+ cDCs was examined in MLR and by CFSE assay. Purified CD4+ T cells were stimulated with splenic DCs (B), and in vitro-generated PIR-A/B+ cDCs were added to MLR (C). Furthermore, anti-IL-27 Ab or anti-TGF-β Ab was added to MLR to assess the effect on on the inhibitory function of PIR-A/B+ cDCs. The number in the figure shows the percentage of cells in each region (means and standard deviations of triplicated cultures. $; p<0.01, **; p<0.05). (JPEG 502 kb)

535_2018_1447_MOESM7_ESM.jpg

Supplementary Figure 6. IL-2 production by CD4+ T cells in MLR. IL-2 in the supernatant of the MLR was determined using an ELISA kit for IL-2. Columns and bars in the figure represent means and standard deviations of triplicated cultures. (JPEG 398 kb)

535_2018_1447_MOESM8_ESM.xls

Footnote for Supplementary Table 1. The expression of cytokine-related genes assayed by a microarray analysis is represented as excel data. (XLS 1444 kb)

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Matsui, F., Inaba, M., Uchida, K. et al. Induction of PIR-A/B+ DCs in the in vitro inflammatory condition and their immunoregulatory function. J Gastroenterol 53, 1131–1141 (2018). https://doi.org/10.1007/s00535-018-1447-1

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  • DOI: https://doi.org/10.1007/s00535-018-1447-1

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