Yoon Seok Choi1, Min Kyung Jung2, Jeewon Lee2, Seong Jin Choi2, Sung Hoon Choi3, Hyun Woong Lee4, Jong-Joo Lee5, Hyung Joon Kim4, Sang Hoon Ahn3, Dong Hyeon Lee6, Won Kim6, Su-Hyung Park7, Jun R Huh8, Hyoung-Pyo Kim5, Jun Yong Park9, Eui-Cheol Shin10. 1. Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea; Department of Internal Medicine, Chungnam National University College of Medicine, Daejeon, Republic of Korea. 2. Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea. 3. Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea. 4. Department of Internal Medicine, Chung-Ang University College of Medicine, Seoul, Republic of Korea. 5. Department of Environmental Medical Biology, Institute of Tropical Medicine, and Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea. 6. Department of Internal Medicine, Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul, Republic of Korea. 7. Laboratory of Translational Immunology and Vaccinology, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea. 8. Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts. 9. Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea. Electronic address: DRPJY@yuhs.ac. 10. Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea. Electronic address: ecshin@kaist.ac.kr.
Abstract
BACKGROUND AND AIMS: CD4+CD25+Foxp3+ T-regulatory (Treg) cells control immune responses and maintain immune homeostasis. However, under inflammatory conditions, Treg cells produce cytokines that promote inflammation. We investigated production of tumor necrosis factor (TNF) by Treg cells in patients with acute hepatitis A (AHA), and examined the characteristics of these cells and association with clinical factors. METHODS: We analyzed blood samples collected from 63 patients with AHA at the time of hospitalization (and some at later time points) and 19 healthy donors in South Korea. Liver tissues were collected from patients with fulminant AHA during liver transplantation. Peripheral blood mononuclear cells were isolated from whole blood and lymphocytes were isolated from liver tissues and analyzed by flow cytometry. Cytokine production from Treg cells (CD4+CD25+Foxp3+) was measured by immunofluorescence levels following stimulation with anti-CD3 and anti-CD28. Epigenetic stability of Treg cells was determined based on DNA methylation patterns. Phenotypes of Treg cells were analyzed by flow cytometry and an RORγt inhibitor, ML-209, was used to inhibit TNF production. Treg cell suppression assay was performed by co-culture of Treg-depleted peripheral blood mononuclear cells s and isolated Treg cells. RESULTS: A higher proportion of CD4+CD25+Foxp3+ Treg cells from patients with AHA compared with controls produced TNF upon stimulation with anti-CD3 and anti-CD28 (11.2% vs 2.8%). DNA methylation analysis confirmed the identity of the Treg cells. TNF-producing Treg cells had features of T-helper 17 cells, including up-regulation of RORγt, which was required for TNF production. The Treg cells had reduced suppressive functions compared with Treg cells from controls. The frequency of TNF-producing Treg cells in AHA patients' blood correlated with their serum level of alanine aminotransferase. CONCLUSIONS: Treg cells from patients with AHA have altered functions compared with Treg cells from healthy individuals. Treg cells from patients with AHA produce higher levels of TNF, gain features of T-helper 17 cells, and have reduced suppressive activity. The presence of these cells is associated with severe liver injury in patients with AHA.
BACKGROUND AND AIMS: CD4+CD25+Foxp3+ T-regulatory (Treg) cells control immune responses and maintain immune homeostasis. However, under inflammatory conditions, Treg cells produce cytokines that promote inflammation. We investigated production of tumor necrosis factor (TNF) by Treg cells in patients with acute hepatitis A (AHA), and examined the characteristics of these cells and association with clinical factors. METHODS: We analyzed blood samples collected from 63 patients with AHA at the time of hospitalization (and some at later time points) and 19 healthy donors in South Korea. Liver tissues were collected from patients with fulminant AHA during liver transplantation. Peripheral blood mononuclear cells were isolated from whole blood and lymphocytes were isolated from liver tissues and analyzed by flow cytometry. Cytokine production from Treg cells (CD4+CD25+Foxp3+) was measured by immunofluorescence levels following stimulation with anti-CD3 and anti-CD28. Epigenetic stability of Treg cells was determined based on DNA methylation patterns. Phenotypes of Treg cells were analyzed by flow cytometry and an RORγt inhibitor, ML-209, was used to inhibit TNF production. Treg cell suppression assay was performed by co-culture of Treg-depleted peripheral blood mononuclear cells s and isolated Treg cells. RESULTS: A higher proportion of CD4+CD25+Foxp3+ Treg cells from patients with AHA compared with controls produced TNF upon stimulation with anti-CD3 and anti-CD28 (11.2% vs 2.8%). DNA methylation analysis confirmed the identity of the Treg cells. TNF-producing Treg cells had features of T-helper 17 cells, including up-regulation of RORγt, which was required for TNF production. The Treg cells had reduced suppressive functions compared with Treg cells from controls. The frequency of TNF-producing Treg cells in AHA patients' blood correlated with their serum level of alanine aminotransferase. CONCLUSIONS: Treg cells from patients with AHA have altered functions compared with Treg cells from healthy individuals. Treg cells from patients with AHA produce higher levels of TNF, gain features of T-helper 17 cells, and have reduced suppressive activity. The presence of these cells is associated with severe liver injury in patients with AHA.
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