Interferon-gamma and tumor necrosis factor-alpha synergistically enhance the immunosuppressive capacity of human umbilical-cord-derived mesenchymal stem cells by increasing PD-L1 expression

Figure 1 Interferon-gamma induced the expression of programmed cell death protein 1 ligand 1 in human umbilical-cord-mesenchymal stem cells. A-C: Programmed cell death protein 1 ligand 1 (PD-L1) expression was measured by quantitative real-time PCR (qRT-PCR), flow cytometry, and western blot in human umbilical-cord-mesenchymal stem cells (hUC-MSCs) treated with 0, 10, 20, 40, 60, 80 or 100 ng/mL interferon-gamma (IFN-γ) for 24 h; D-F: PD-L1 expression was measured by qRT-PCR, flow cytometry, and western blot in hUC-MSCs treated with 20 ng/mL IFN-γ for 0, 2, 4, 6, 8, 12, 24 or 48 h. Data were represented as mean ± SEM of n = 3. Iso: Isotype; C: Control; ^aP < 0.01; ^bP < 0.001. IFN-γ: Interferon-gamma; PD-L1: Programmed cell death protein 1 ligand 1.

Figure 2 Interferon-gamma upregulated programmed cell death protein 1 ligand 1 expression via the JAK/STAT1/ interferon regulatory factor 1 pathway in human umbilical-cord-mesenchymal stem cells. A: Programmed cell death protein 1 ligand 1 (PD-L1) expression was quantified by quantitative real-time PCR (qRT-PCR) in human umbilical-cord-mesenchymal stem cells (hUC-MSCs) treated with a combination of IFN-γ with or without the JAK inhibitor ruxolitinib (10 µM) for 4 h; B: PD-L1 expression was quantified by flow cytometry in hUC-MSCs treated with a combination of IFN-γ with or without ruxolitinib (10 M) for 24 h; C: Activation of the JAK/STAT/interferon regulatory factor 1 (IRF1) pathway and PD-L1 expression were analyzed by western blot in hUC-MSCs treated with IFN-γ (20 ng/mL) with or without ruxolitinib (10 M) for 24 h; D: The PD-L1 expression was quantified by qRT-PCR in hUC-MSCs transfected with siRNA against JAK1, JAK2, STAT1, STAT2, STAT3, IRF1, or IRF9 and subsequently treated with IFN-γ for 4 h. The expression of PD-L1 in hUC-MSCs of the IFN-γ + si-NC (negative control) group was used as the standard (100%); E: PGL4.10-PD-L1 promoter, pIRES2-IRF1, and Renilla luciferase plasmids were transfected together or separately into 293T cells. Cells were lysed 24 h after transfection and luciferase activity was measured; F: pIRES2-IRF1 plasmid and Renilla luciferase plasmid were transfected into 293T cells with the PGL4.10-PD-L1 promoter plasmid or IRF1 binding site mutant PGL4.10-PD-L1 promoter plasmids. Cells were lysed 24 h after transfection and luciferase activity was measured; G: Predicted binding sites of IRF1 on the PD-L1 promoter. Data were represented as mean ± SEM of n = 3. C: Control; Iso: Isotype; ^aP < 0.01; ^cP < 0.0001. IFN-γ: Interferon-gamma; PD-L1: Programmed cell death protein 1 ligand 1.

Figure 3 Tumor necrosis factor-alpha synergistically enhanced interferon-gamma-induced programmed cell death protein 1 ligand 1 expression in human umbilical-cord-mesenchymal stem cells. A: Programmed cell death protein 1 ligand 1 (PD-L1) expression was measured by flow cytometry in human umbilical-cord-mesenchymal stem cells (hUC-MSCs) treated with 0, 10, 20, 40, 60, 80 or 100 ng/mL tumor necrosis factor-alpha (TNF-α) for 24 h; B: PD-L1 expression was measured by flow cytometry in hUC-MSCs treated with 100 ng/mL TNF-α for 0, 2, 4, 6, 8, 12, 24, or 48 h; C: Proliferation was measured by CCK8 in hUC-MSCs treated with 0, 10, 20, 40, 60, 80 or 100 ng/mL of TNF-α for 24 h; D: Apoptotic hUC-MSCs were measured by flow cytometry in hUC-MSCs treated with 0, 10, 20, 40, 60, 80 or 100 ng/mL TNF-α for 24 h. The histogram shows the quantification results pertaining to the percentage of apoptotic cells (Annexin V⁺ cells) in each group; E: PD-L1 expression was quantified by flow cytometry in hUC-MSCs after 24 h of incubation with IFN-γ (20 ng/mL) and 0, 10, 20, 40, 60, 80 or 100 ng/mL TNF-α; F: PD-L1 expression was measured by quantitative real-time PCR in hUC-MSCs treated with IFN-γ (20 ng/mL), TNF-α (10 ng/mL), or a combination of IFN-γ (20 ng/mL) and TNF-α (10 ng/mL) for 4 h; G: PD-L1 expression was analyzed by western blot in hUC-MSCs treated with IFN-γ (20 ng/mL), TNF-α (10 ng/mL), or a combination of IFN-γ (20 ng/mL) and TNF-α (10 ng/mL) for 24 h. Data were represented as mean ± SEM of n = 3. C: Control; Iso: Isotype; ^bP < 0.001; ^cP < 0.0001; ^dP > 0.05. IFN-γ: Interferon-gamma; PD-L1: Programmed cell death protein 1 ligand 1; TNF-α: Tumor necrosis factor-alpha.

Figure 4 Tumor necrosis factor-alpha increased programmed cell death protein 1 ligand expression by promoting interferon-gamma-induced activation of the JAK/STAT1/interferon regulatory factor 1 pathway. A: Programmed cell death protein 1 ligand (PD-L1) expression was quantified by quantitative real-time PCR (qRT-PCR) in human umbilical-cord-mesenchymal stem cells (hUC-MSCs) after 4 h of incubation with IFN-γ and tumor necrosis factor-alpha (TNF-α) in the presence or absence of ruxolitinib (10 μM) or the NF-κB inhibitor BAY11-7082 (10 μM); B: PD-L1 expression in hUC-MSCs was quantified by flow cytometry after 24 h of incubation with IFN-γ and TNF-α in the presence or absence of ruxolitinib (10 μM); C: Activation of the JAK/STAT1/interferon regulatory factor 1 (IRF1) pathway and the expression of PD-L1 were analyzed by western blot in hUC-MSCs after 24 h of incubation with IFN-γ and TNF-α in the presence or absence of ruxolitinib (10 μM); D and E: PD-L1 expression was quantified by quantitative real-time PCR and flow cytometry in hUC-MSCs transfected with siRNA of NC, STAT1, or IRF1 and subsequently treated with IFN-γ and TNF-α for 4 h or 24 h; F: Activation of the JAK/STAT1/IRF1 pathway was analyzed by western blot in hUC-MSCs treated with IFN-γ (20 ng/mL), TNF-α (10 ng/mL), or a combination of IFN-γ (20 ng/mL) and TNF-α (10 ng/mL) for 24 h. Data were represented as mean ± SEM of n = 3. C: Control; Iso: Isotype; ^cP < 0.0001. IFN-γ: Interferon-gamma; PD-L1: Programmed cell death protein 1 ligand 1; TNF-α: Tumor necrosis factor-alpha.

Figure 5 Tumor necrosis factor-alpha synergistically amplified interferon-gamma-induced JAK/STAT1/interferon regulatory factor 1 signaling activation by upregulating NF-κB-mediated interferon-gamma receptor 1/2 expression. A: The expression of NF-κB-mediated interferon-gamma receptor 1/2 (IFNGR1/2) were quantified by quantitative real-time PCR (qRT-PCR) in human umbilical-cord-mesenchymal stem cells (hUC-MSCs) treated with IFN-γ and tumor necrosis factor-alpha (TNF-α) alone or in combination for 4 h; B and C: IFNGR1 expression was quantified by flow cytometry or WB in hUC-MSCs treated with IFN-γ and TNF-α alone or in combination for 24 h; D and E: PD-L1 expression was quantified by qRT-PCR or flow cytometry in hUC-MSCs transfected with siRNA against NC, IFNGR1, or IFNGR2, and subsequently treated with IFN-γ and TNF-α for 4 h or 24 h; F: Activation of the JAK/STAT1/interferon regulatory factor 1 pathway and the expressions of PD-L1 and IFNGR1 were analyzed by western blot in hUC-MSCs after 24 h of incubation with IFN-γ and TNF-α in the presence or absence of IFNGR1 siRNA; G: Activation of the NF-κB pathway and expression of IFNGR1 were analyzed by western blot in hUC-MSCs after 24 h of incubation with TNF-α in the presence or absence of the NF-KB inhibitor BAY11-7082 (10 μM); H: IKKα, IKKβ, p65 and IκBα protein levels in the cytoplasm and nucleus of hUC-MSCs after TNF-α stimulation were measured through western blot; I: IFNGR1 expression was quantified by qRT-PCR in hUC-MSCs transfected with p65 siRNA and subsequently treated with IFN-γ and TNF-α for 4 h; J and K: PD-L1 expression was quantified by qRT-PCR or flow cytometry in hUC-MSCs transfected with p65 siRNA and subsequently treated with IFN-γ and TNF-α for 4 h or 24 h; L: hUC-MSCs were infected with IFNGR1 overexpressed lentivirus and transfected with siRNA of p65, and induced with IFN-γ and TNF-α for 4 h, and then PD-L1 expression was quantified by qRT-PCR; M: PGL4.10-IFNGR1 promoter plasmid, pIRES2-p65 plasmid, and Renilla luciferase plasmid were transfected together or separately into 293T cells. Cells were lysed 24 h after transfection and luciferase activity was measured; N: pIRES2-p65 plasmid and Renilla luciferase plasmid were transfected into 293T cells with the PGL4.10-IFNGR1 promoter plasmid or the p65 binding site mutant PGL4.10-IFNGR1 promoter plasmid. Cells were lysed 24 h after transfection and luciferase activity was measured. The predicted binding sites of p65 to the IFNGR1 promoter are above the graph. Data were represented as mean ± SEM of n = 3. C: Control; Iso: Isotype; ^bP < 0.001; ^cP < 0.0001; ^dP > 0.05. IFN-γ: Interferon-gamma; PD-L1: Programmed cell death protein 1 ligand 1; TNF-α: Tumor necrosis factor-alpha.

Figure 6 Synergy by IFN-γ and tumor necrosis factor-alpha enhanced the immunosuppressive capacity of human umbilical-cord-mesenchymal stem cells by inducing programmed cell death protein 1 ligand expression. A: The CFSE fluorescence intensity of CFSE-labeled peripheral blood mononuclear cells (PBMCs) was detected after co-culture with human umbilical-cord-mesenchymal stem cells (hUC-MSCs) with different treatments for 3 d. The CFSE fluorescence intensity of CD3-positive PBMCs on day 0 was used as the reference (1.29%). The histogram shows the quantification results pertaining to the mean fluorescent intensity of CFSE in each group; B: Schematic diagram of dextran sulfate sodium-induced colitis and MSCs transplantation therapy in mice; C: Bodyweight of mice in each group during the disease process (n = 6); D: Macroscopic appearance of the colon in each group; E: Colon sections embedded in paraffin were stained with hematoxylin and eosin for light microscopy assessment. Scale bar, 50 μm; F: Histopathological score of colons from each group was assessed for disease severity (n = 3); G: Infiltration cells expressing CD4 or CD11b in colon tissue sections from each group were analyzed by immunohistochemical staining (n = 3). Scale bar, 50 μm; H: Myeloperoxidase activity in the colon was determined (n = 3); I: The colonic mRNA expression of Treg (Foxp3) from each group were analyzed by quantitative real-time PCR (n = 3); J: Levels of inflammatory factors in colon tissue homogenates from each group were determined using ELISA (n = 3). IT-MSCs refer to hUC-MSCs pretreated with IFN-γ and tumor necrosis factor-alpha for 24 h. C: Control; ^aP < 0.01; ^bP < 0.001; ^cP < 0.0001; ^dP > 0.05.

Figure 7 Molecular mechanism of interferon-gamma and tumor necrosis factor-alpha synergistically upregulated expression of programmed cell death protein 1 ligand in human umbilical-cord-mesenchymal stem cells to inhibit T cells. IFN-γ: Interferon-gamma; PD-L1: Programmed cell death protein 1 ligand 1; TNF-α: Tumor necrosis factor-alpha; hUC-MSCs: Human umbilical-cord-mesenchymal stem cells; IRF1: Interferon regulatory factor 1.