Immune Netw.  2015 Oct;15(5):241-251. 10.4110/in.2015.15.5.241.

Galectin-9 is Involved in Immunosuppression Mediated by Human Bone Marrow-derived Clonal Mesenchymal Stem Cells

Affiliations
  • 1Drug Development Program, Department of Medicine, Inha University School of Medicine, Incheon 22332, Korea.
  • 2Translational Research Center, Inha University School of Medicine, Incheon 22332, Korea. sunuksong@inha.ac.kr, tgwise@inha.ac.kr
  • 3Inha Research Institute for Medical Sciences of Biomedical Sciences, Inha University School of Medicine, Incheon 22332, Korea.
  • 4SCM Lifescience Co. Ltd., Incheon 22332, Korea.

Abstract

Bone marrow-derived mesenchymal stem cells (MSCs) have immunomodulatory properties and can suppress exaggerated pro-inflammatory immune responses. Although the exact mechanisms remain unclear, a variety of soluble factors are known to contribute to MSC-mediated immunosuppression. However, functional redundancy in the immunosuppressive properties of MSCs indicates that other uncharacterized factors could be involved. Galectin-9, a member of the beta-galactoside binding galectin family, has emerged as an important regulator of innate and adaptive immunity. We examined whether galectin-9 contributes to MSC-mediated immunosuppression. Galectin-9 was strongly induced and secreted from human MSCs upon stimulation with pro-inflammatory cytokines. An in vitro immunosuppression assay using a knockdown approach revealed that galectin-9-deficient MSCs do not exert immunosuppressive activity. We also provided evidence that galectin-9 may contribute to MSC-mediated immunosuppression by binding to its receptor, TIM-3, expressed on activated lymphocytes, leading to apoptotic cell death of activated lymphocytes. Taken together, our findings demonstrate that galectin-9 is involved in MSC-mediated immunosuppression and represents a potential therapeutic factor for the treatment of inflammatory diseases.

Keyword

Human mesenchymal stem cells; Immunosuppression; Galectin-9; Apoptosis; TIM-3

MeSH Terms

Adaptive Immunity
Apoptosis
Cell Death
Cytokines
Galectins
Humans*
Immunosuppression*
Lymphocytes
Mesenchymal Stromal Cells*
Cytokines
Galectins

Figure

  • Figure 1 Galectin-9 induction in MSCs by experimental inflammatory stimulation. (A) MSCs were stimulated with 10 ng/ml TNF-α and 20 ng/ml IFN-γ for 24 h and expression of IDO, galectin-1, galectin-9 and GAPDH was detected by RT-PCR. (B) After MSCs were stimulated with 10 ng/ml TNF-α and 20 ng/ml IFN-γ for 24 and 48 h, flow cytometric analysis was conducted to detect intracellular expression of galectin-9. Red lines histograms indicate intracellular galectin-9 after stimulation while black-filled histograms represent unstimulated controls. (C) MSCs were stimulated with 10 ng/ml TNF-α and 20 ng/ml IFN-γ up to 72 h and then secreted galectin-9 in the culture supernatant was quantified by ELISA. Data represent the average of three independent experiments.

  • Figure 2 The immunosuppression of galectin-9 or MSCs on human lymphocyte proliferation. (A) In vitro immunosuppressive effects of galectin-9 or MSCs on human lymphocyte proliferation were evaluated by using mitogenically activated human PBMCs (P). PBMCs stimulated with PHA (1 µg/ml) were treated with 1.5 µg/ml of recombinant human galectin-9 (rhGal-9) or co-cultured with MSCs (the cell number ratio of MSC to PBMC is 1:5) for 3 days. Lymphocyte proliferation was determined by [3H]-thymidine incorporation. (B) CFSE-labeled PBMCs stimulated with PHA were incubated with 1.5 µg/ml rhGal-9 or co-cultured with MSCs (MSC:PBMC=1:5) for 5 days followed by flow cytometric analysis for cell division. Dividing PBMCs were shown in percentage. Data shown are representative of three independent experiments. (C) Galectin-9, TNF-α, and IL-17A in the culture supernatants were quantitated by ELISA. Data are the average of three independent experiments. Statistical significance was *p<0.05 or **p<0.005. P: PBMC.

  • Figure 3 The effects of galectin-9-knockdown MSCs on proliferation of activated lymphocytes. (A) MSCs were transfected with siRNA oligomers specific to galectin-9 (siGal-9) or scrambled control oligomers (siCon). After the cells were stimulated with 10 ng/ml TNF-α and 20 ng/ml IFN-γ for 48 h, western blot analysis was performed to check galectin-9 knockdown. (B) siGal-9-transfected MSCs were stimulated with10 ng/ml TNF-α and 20 ng/ml IFN-γ for 48 h and then secretion of galectin-9 was determined by ELISA. (C) PBMCs stimulated with 1 µg/ml PHA were co-cultured with siCon- or siGal-9-transfected MSCs (MSC:PBMC=1:5) for 3 days. Lymphocyte proliferation was determined by [3H]-thymidine incorporation. Galectin-9 knockdown by siGal-9-transfection abrogated the immunosuppressive activity of MSCs. Data shown are the average of three independent experiments. (D) MSCs were infected with shRNA-expressing lentivirus targeting galectin-9 (shGal-9) or control LacZ virus (shLacZ). After the cells were stimulated with 10 ng/ml TNF-α and 20 ng/ml IFN-γ for 48 h, western blot analysis was performed to check galectin-9 knockdown. (E) The MSCs were stimulated with 10 ng/ml TNF-α and 20 ng/ml IFN-γ for 48 h and then soluble galectin-9 released into the culture supernatant was determined by ELISA. (F) PBMCs stimulated with 1 µg/ml PHA were co-cultured with shLacZ- or shGal-9-infected MSCs for 3 days. Lymphocyte proliferation was determined as in (C). MSC-suppressed proliferation of activated PBMCs was significantly recovered in the co-culture of shGal-9-transfected MSCs. Data are the average of three independent experiments. Statistical significance was *p< 0.05 or **p<0.005. P: PBMC.

  • Figure 4 The effects of galectin-9 or MSCs on TIM-3-expressing lymphocytes and apoptotic analysis of activated lymphocytes by galectin-9-or MSCs. (A) After PHA-activated PBMCs were treated with exogenous rhGal-9 at a concentration of 0.3 or 1.5 µg/ml or were co-cultured with MSCs at the indicated cell ratios for 3 days, flow cytometric analysis was conducted to examine the effects of galectin-9 or MSCs on TIM-3-expressing CD3+ lymphocytes. (B) The apoptosis-inducing effects of galectin-9 or MSCs on PHA-activated PBMCs were evaluated. In the presence of rhGal-9 or MSCs, activation-induced cell death of PBMCs was significantly increased. Apoptotic cell death induced by rhGal-9 was comparable to that induced by MSCs. Apoptotic cell death was evaluated by annexin V and PI staining. Data are the average of three independent experiments. Statistical significance was *p<0.05 or **p<0.005. P: PBMC.


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