Abstract
Background: For xenotransplantation to truly succeed, we must develop immunomodulatory strategies to suppress the xenoimmune response but by minimizing immunosuppression over the long term. Regulatory macrophages (Mreg) have been shown to suppress polyclonal T-cell proliferation in vitro and prolong allograft survival in vivo. However, the question of whether they are capable of suppressing xenoimmune responses remains unknown. This study assessed the potential of human Mreg to be used as an effective immunomodulatory method in xenotransplantation. Methods: CD14+ monocytes selected from human peripheral blood mononuclear cells (PBMC) were cultured with macrophage colony-stimulating factor (M-CSF) for 7 days with IFN-γ added at day 6 for Mreg induction. Mreg phenotyping was performed by flow cytometric analysis, and the in vitro suppressive function was assessed by mixed lymphocyte reaction (MLR) using irradiated pig PBMC as the xenogeneic stimulator cells, human PBMC as responder cells, and autologous Mreg as suppressor cells. To assess mRNA expression of Mreg functional molecules indoleamine-2,3-dioxygenase (IDO), IL-10, inducible nitric oxide synthase (iNOS) and TGF-β were measured by real-time PCR. Supernatants were collected from the MLR cultures for IDO activity assay by high-performance liquid chromatography (HPLC). The effects of the IDO inhibitor 1-D/L-methyl-tryptophan (1-MT), iNOS inhibitor NG-monomethyl-l-arginine (L-NMMA), and anti-IFN-γ or anti-TGF-β monoclonal antibody (mAb) treatment on Mreg suppressive capacity were tested from the supernatants of the MLR assays. Results: We demonstrated that induced Mreg with a phenotype of CD14lowCD16-/lowCD80lowCD83-/lowCD86+/hiHLA-DR+/hi were capable of suppressing proliferating human PBMC, CD4+, and CD8+ T cells, even at a higher responder:Mreg ratio of 32:1 in a pig-human xenogeneic MLR. The strong suppressive potency of Mreg was further correlated with their upregulated IDO expression and activity. The IDO upregulation of Mreg was associated with an increased production of IFN-γ, an IDO stimulator, by xenoreactive responder cells in the xenogeneic MLR. While no effect on Mreg suppressive potency was detected by addition of the iNOS inhibitor L-NMMA or anti-TGF-β mAb into the MLR assays, inhibition of IDO activity by neutralizing IFN-γ or by IDO inhibitor 1-MT substantially impaired the capacity of Mreg to suppress the xenogeneic response, indicating the importance of upregulated IDO activity in Mreg-mediated suppression of the xenogeneic response in vitro. Conclusion: This study demonstrates that human Mreg are capable of suppressing the xenoimmune response in vitro via IDO-involved mechanism(s), suggesting their potential role as an effective immunomodulatory tool in xenotransplantation.
| Language | English |
|---|---|
| Article number | e12326 |
| Pages | 1-11 |
| Number of pages | 11 |
| Journal | Xenotransplantation |
| Volume | 24 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - Oct 2017 |
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Human regulatory macrophages are potent in suppression of the xenoimmune response via indoleamine-2,3-dioxygenase-involved mechanism(s). / Guo, Fei; Hu, Min; Huang, Dandan; Zhao, Yuanfei; Heng, Benjamin; Guillemin, Gilles; Lim, Chai K.; Hawthorne, Wayne J.; Yi, Shounan.
In: Xenotransplantation, Vol. 24, No. 5, e12326, 10.2017, p. 1-11.Research output: Contribution to journal › Article › Research › peer-review
TY - JOUR
T1 - Human regulatory macrophages are potent in suppression of the xenoimmune response via indoleamine-2,3-dioxygenase-involved mechanism(s)
AU - Guo,Fei
AU - Hu,Min
AU - Huang,Dandan
AU - Zhao,Yuanfei
AU - Heng,Benjamin
AU - Guillemin,Gilles
AU - Lim,Chai K.
AU - Hawthorne,Wayne J.
AU - Yi,Shounan
PY - 2017/10
Y1 - 2017/10
N2 - Background: For xenotransplantation to truly succeed, we must develop immunomodulatory strategies to suppress the xenoimmune response but by minimizing immunosuppression over the long term. Regulatory macrophages (Mreg) have been shown to suppress polyclonal T-cell proliferation in vitro and prolong allograft survival in vivo. However, the question of whether they are capable of suppressing xenoimmune responses remains unknown. This study assessed the potential of human Mreg to be used as an effective immunomodulatory method in xenotransplantation. Methods: CD14+ monocytes selected from human peripheral blood mononuclear cells (PBMC) were cultured with macrophage colony-stimulating factor (M-CSF) for 7 days with IFN-γ added at day 6 for Mreg induction. Mreg phenotyping was performed by flow cytometric analysis, and the in vitro suppressive function was assessed by mixed lymphocyte reaction (MLR) using irradiated pig PBMC as the xenogeneic stimulator cells, human PBMC as responder cells, and autologous Mreg as suppressor cells. To assess mRNA expression of Mreg functional molecules indoleamine-2,3-dioxygenase (IDO), IL-10, inducible nitric oxide synthase (iNOS) and TGF-β were measured by real-time PCR. Supernatants were collected from the MLR cultures for IDO activity assay by high-performance liquid chromatography (HPLC). The effects of the IDO inhibitor 1-D/L-methyl-tryptophan (1-MT), iNOS inhibitor NG-monomethyl-l-arginine (L-NMMA), and anti-IFN-γ or anti-TGF-β monoclonal antibody (mAb) treatment on Mreg suppressive capacity were tested from the supernatants of the MLR assays. Results: We demonstrated that induced Mreg with a phenotype of CD14lowCD16-/lowCD80lowCD83-/lowCD86+/hiHLA-DR+/hi were capable of suppressing proliferating human PBMC, CD4+, and CD8+ T cells, even at a higher responder:Mreg ratio of 32:1 in a pig-human xenogeneic MLR. The strong suppressive potency of Mreg was further correlated with their upregulated IDO expression and activity. The IDO upregulation of Mreg was associated with an increased production of IFN-γ, an IDO stimulator, by xenoreactive responder cells in the xenogeneic MLR. While no effect on Mreg suppressive potency was detected by addition of the iNOS inhibitor L-NMMA or anti-TGF-β mAb into the MLR assays, inhibition of IDO activity by neutralizing IFN-γ or by IDO inhibitor 1-MT substantially impaired the capacity of Mreg to suppress the xenogeneic response, indicating the importance of upregulated IDO activity in Mreg-mediated suppression of the xenogeneic response in vitro. Conclusion: This study demonstrates that human Mreg are capable of suppressing the xenoimmune response in vitro via IDO-involved mechanism(s), suggesting their potential role as an effective immunomodulatory tool in xenotransplantation.
AB - Background: For xenotransplantation to truly succeed, we must develop immunomodulatory strategies to suppress the xenoimmune response but by minimizing immunosuppression over the long term. Regulatory macrophages (Mreg) have been shown to suppress polyclonal T-cell proliferation in vitro and prolong allograft survival in vivo. However, the question of whether they are capable of suppressing xenoimmune responses remains unknown. This study assessed the potential of human Mreg to be used as an effective immunomodulatory method in xenotransplantation. Methods: CD14+ monocytes selected from human peripheral blood mononuclear cells (PBMC) were cultured with macrophage colony-stimulating factor (M-CSF) for 7 days with IFN-γ added at day 6 for Mreg induction. Mreg phenotyping was performed by flow cytometric analysis, and the in vitro suppressive function was assessed by mixed lymphocyte reaction (MLR) using irradiated pig PBMC as the xenogeneic stimulator cells, human PBMC as responder cells, and autologous Mreg as suppressor cells. To assess mRNA expression of Mreg functional molecules indoleamine-2,3-dioxygenase (IDO), IL-10, inducible nitric oxide synthase (iNOS) and TGF-β were measured by real-time PCR. Supernatants were collected from the MLR cultures for IDO activity assay by high-performance liquid chromatography (HPLC). The effects of the IDO inhibitor 1-D/L-methyl-tryptophan (1-MT), iNOS inhibitor NG-monomethyl-l-arginine (L-NMMA), and anti-IFN-γ or anti-TGF-β monoclonal antibody (mAb) treatment on Mreg suppressive capacity were tested from the supernatants of the MLR assays. Results: We demonstrated that induced Mreg with a phenotype of CD14lowCD16-/lowCD80lowCD83-/lowCD86+/hiHLA-DR+/hi were capable of suppressing proliferating human PBMC, CD4+, and CD8+ T cells, even at a higher responder:Mreg ratio of 32:1 in a pig-human xenogeneic MLR. The strong suppressive potency of Mreg was further correlated with their upregulated IDO expression and activity. The IDO upregulation of Mreg was associated with an increased production of IFN-γ, an IDO stimulator, by xenoreactive responder cells in the xenogeneic MLR. While no effect on Mreg suppressive potency was detected by addition of the iNOS inhibitor L-NMMA or anti-TGF-β mAb into the MLR assays, inhibition of IDO activity by neutralizing IFN-γ or by IDO inhibitor 1-MT substantially impaired the capacity of Mreg to suppress the xenogeneic response, indicating the importance of upregulated IDO activity in Mreg-mediated suppression of the xenogeneic response in vitro. Conclusion: This study demonstrates that human Mreg are capable of suppressing the xenoimmune response in vitro via IDO-involved mechanism(s), suggesting their potential role as an effective immunomodulatory tool in xenotransplantation.
KW - Indoleamine-2,3-dioxygenase
KW - Regulatory macrophages
KW - Transplantation tolerance
KW - Xenotransplantation
UR - http://www.scopus.com/inward/record.url?scp=85026664020&partnerID=8YFLogxK
UR - https://ulrichsweb.serialssolutions.com/title/1505879644016/235378
U2 - 10.1111/xen.12326
DO - 10.1111/xen.12326
M3 - Article
VL - 24
SP - 1
EP - 11
JO - Xenotransplantation
T2 - Xenotransplantation
JF - Xenotransplantation
SN - 0908-665X
IS - 5
M1 - e12326
ER -