Cheuk-Lun Lee1, YiFan Guo2, Kam-Hei So3, Madhavi Vijayan3, Yue Guo3, Vera H H Wong3, YuanQing Yao4, Kai-Fai Lee1, Philip C N Chiu1, William S B Yeung5. 1. Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam Road, Hong Kong, China Centre for Reproduction, Development and Growth, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam Road, Hong Kong, China Shenzhen Key Laboratory of Fertility Regulation, Department of Obstetrics and Gynecology, The University of Hong Kong-Shenzhen Hospital, Shenzhen, China. 2. Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam Road, Hong Kong, China Department of Obstetrics and Gynaecology, Chinese PLA General Hospital, 28 Fuxing Road, Beijing, China. 3. Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam Road, Hong Kong, China. 4. Department of Obstetrics and Gynaecology, Chinese PLA General Hospital, 28 Fuxing Road, Beijing, China. 5. Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam Road, Hong Kong, China Centre for Reproduction, Development and Growth, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam Road, Hong Kong, China Shenzhen Key Laboratory of Fertility Regulation, Department of Obstetrics and Gynecology, The University of Hong Kong-Shenzhen Hospital, Shenzhen, China wsbyeung@hku.hk.
Abstract
STUDY QUESTION: What are the actions of soluble human leukocyte antigen G5 (sHLAG5) on macrophage differentiation? SUMMARY ANSWER: sHLAG5 polarizes the differentiation of macrophages toward a decidual macrophage-like phenotype, which could regulate fetomaternal tolerance and placental development. WHAT IS KNOWN ALREADY: sHLAG5 is a full-length soluble isoform of human leukocyte antigen implicated in immune tolerance during pregnancy. Low or undetectable circulating level of sHLAG5 in first trimester of pregnancy is associated with pregnancy complications such as pre-eclampsia and spontaneous abortion. Decidual macrophages are located in close proximity to invasive trophoblasts, and are involved in regulating fetomaternal tolerance and placental development. STUDY DESIGN, SIZE, DURATION: Human peripheral blood monocytes were differentiated into macrophages by treatment with granulocyte macrophage colony-stimulating factor in the presence or absence of recombinant sHLAG5 during the differentiation process. The phenotypes and the biological activities of the resulting macrophages were compared. PARTICIPANTS/MATERIALS, SETTING, METHODS: Recombinant sHLAG5 was produced in Escherichia coli BL21 and the protein identity was verified by tandem mass spectrometry. The expression of macrophage markers were analyzed by flow cytometry and quantitative PCR. Phagocytosis was determined by flow cytometry. Indoleamine 2,3-dioxygenase 1 expression and activity were measured by western blot analysis and kynurenine assay, respectively. Cell proliferation and cell cycling were determined by fluorometric cell proliferation assay and flow cytometry, respectively. Cytokine secretion was determined by cytokine array and ELISA kits. Intracellular cytokine expression was measured by flow cytometry. Cell invasion and migration were determined by trans-well invasion and migration assay, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: sHLAG5 drove the differentiation of macrophages with 'immuno-modulatory' characteristics, including reduced expression of M1 macrophage marker CD86 and increased expression of M2 macrophage marker CD163. sHLAG5-polarized macrophages showed enhanced phagocytic activity. They also had higher expression and activity of indoleamine 2,3-dioxygenase 1, a phenotypic marker of decidual macrophages, which inhibited proliferation of autologous T-cells via induction of G0/G1 cell cycle arrest. In addition, sHLAG5-polarized macrophages had an increased secretion of interleukin-6 and C-X-C motif ligand 1, which inhibited interferon-γ production in T-cells and induction of trophoblast invasion, respectively. LIMITATIONS, REASONS FOR CAUTION: Most information on the phenotypes and biological activities of human decidual macrophages are based on past literatures. A direct comparison between sHLAG5-polarized macrophages and primary decidual macrophages is required to verify the present observations. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study on the role of sHLAG5 in macrophage differentiation. Further study on the mechanism that regulates the differentiation process of macrophages would enhance our understanding on the physiology of early pregnancy. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by the Hong Kong Research Grant Council Grant HKU774212 and the University of Hong Kong Grant 201309176126. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: Nil.
STUDY QUESTION: What are the actions of soluble human leukocyte antigen G5 (sHLAG5) on macrophage differentiation? SUMMARY ANSWER: sHLAG5 polarizes the differentiation of macrophages toward a decidual macrophage-like phenotype, which could regulate fetomaternal tolerance and placental development. WHAT IS KNOWN ALREADY: sHLAG5 is a full-length soluble isoform of human leukocyte antigen implicated in immune tolerance during pregnancy. Low or undetectable circulating level of sHLAG5 in first trimester of pregnancy is associated with pregnancy complications such as pre-eclampsia and spontaneous abortion. Decidual macrophages are located in close proximity to invasive trophoblasts, and are involved in regulating fetomaternal tolerance and placental development. STUDY DESIGN, SIZE, DURATION: Human peripheral blood monocytes were differentiated into macrophages by treatment with granulocyte macrophage colony-stimulating factor in the presence or absence of recombinant sHLAG5 during the differentiation process. The phenotypes and the biological activities of the resulting macrophages were compared. PARTICIPANTS/MATERIALS, SETTING, METHODS: Recombinant sHLAG5 was produced in Escherichia coli BL21 and the protein identity was verified by tandem mass spectrometry. The expression of macrophage markers were analyzed by flow cytometry and quantitative PCR. Phagocytosis was determined by flow cytometry. Indoleamine 2,3-dioxygenase 1 expression and activity were measured by western blot analysis and kynurenine assay, respectively. Cell proliferation and cell cycling were determined by fluorometric cell proliferation assay and flow cytometry, respectively. Cytokine secretion was determined by cytokine array and ELISA kits. Intracellular cytokine expression was measured by flow cytometry. Cell invasion and migration were determined by trans-well invasion and migration assay, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: sHLAG5 drove the differentiation of macrophages with 'immuno-modulatory' characteristics, including reduced expression of M1 macrophage marker CD86 and increased expression of M2 macrophage marker CD163. sHLAG5-polarized macrophages showed enhanced phagocytic activity. They also had higher expression and activity of indoleamine 2,3-dioxygenase 1, a phenotypic marker of decidual macrophages, which inhibited proliferation of autologous T-cells via induction of G0/G1 cell cycle arrest. In addition, sHLAG5-polarized macrophages had an increased secretion of interleukin-6 and C-X-C motif ligand 1, which inhibited interferon-γ production in T-cells and induction of trophoblast invasion, respectively. LIMITATIONS, REASONS FOR CAUTION: Most information on the phenotypes and biological activities of human decidual macrophages are based on past literatures. A direct comparison between sHLAG5-polarized macrophages and primary decidual macrophages is required to verify the present observations. WIDER IMPLICATIONS OF THE FINDINGS: This is the first study on the role of sHLAG5 in macrophage differentiation. Further study on the mechanism that regulates the differentiation process of macrophages would enhance our understanding on the physiology of early pregnancy. STUDY FUNDING/COMPETING INTERESTS: This work was supported in part by the Hong Kong Research Grant Council Grant HKU774212 and the University of Hong Kong Grant 201309176126. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: Nil.