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, 12 (2), 315-330

A Novel Immunomodulatory Mechanism Dependent on Acetylcholine Secreted by Human Bone Marrow-derived Mesenchymal Stem Cells

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A Novel Immunomodulatory Mechanism Dependent on Acetylcholine Secreted by Human Bone Marrow-derived Mesenchymal Stem Cells

Tac-Ghee Yi et al. Int J Stem Cells.

Abstract

Background and objectives: Mesenchymal stem cells (MSCs) are used to treat autoimmune or inflammatory diseases. Our aim was to determine the immunomodulatory mechanisms elicited by MSCs during inflammation.

Methods and results: We cocultured MSCs with peripheral blood mononuclear cells for a mixed lymphocyte reaction or stimulated them by phytohemagglutinin. Morphological changes of MSCs and secretion of acetylcholine (ACh) from MSCs were measured. The effects of an ACh antagonist and ACh agonist on lymphocyte proliferation and proinflammatory-cytokine production were determined. The inflammatory milieu created by immune-cell activation caused MSCs to adopt a neuronlike phenotype and induced them to release ACh. Additionally, nicotinic acetylcholine receptors (nAChRs) were upregulated in activated peripheral blood mononuclear cells. We observed that ACh bound to nAChR on activated immune cells and led to the inhibition of lymphocyte proliferation and of proinflammatory-cytokine production. MSC-mediated immunosuppression through ACh activity was reversed by an ACh antagonist called α-bungarotoxin, and lymphocyte proliferation was inhibited by an ACh agonist, ACh chloride.

Conclusions: Our findings point to a novel immunomodulatory mechanism in which ACh secreted by MSCs under inflammatory conditions might modulate immune cells. This study may provide a novel method for the treatment of autoimmune diseases by means of MSCs.

Keywords: Acetylcholine; Autoimmune diseases; Mesenchymal stem cells; Stem cell therapeutics.

Conflict of interest statement

Potential Conflict of Interest

The authors have no conflicting financial interest.

Figures

Fig. 1
Fig. 1
Inflammatory conditions induce neuronlike morphological features in MSCs. (a) When cocultured in an MLR with PBMCs obtained from two individuals (P and Po), hBM MSCs suppressed the lymphocyte proliferation. (b) When cocultured with human PBMCs activated by PHA stimulation (PPHA), MSCs inhibited the lymphocyte proliferation. CPM: counts per million. (c) Morphological changes in MSCs were observed in cocultures with activated PBMCs. (d, e) Neuronlike morphological changes in MSCs after coculture with activated PBMCs for 48 h. (f, g) Floating neurosphere-like cell clusters (red arrows) were observed under both inflammatory conditions. Error bars are indicative of standard deviations.
Fig. 2
Fig. 2
Inflammation induces the expression of NRs and neuron- or NPC-related markers but not glial markers on the MSC surface. (a) To determine whether inflammation-induced neuronlike phenotypic changes in MSCs were mediated by soluble factors, we performed assays using Transwell plates with inserts (0.4 μm pore size). MSCs (105) were seeded in the bottom well, and MLR-activated PBMCs (106) were seeded in the insert-containing well. After incubation for 3 days, MSCs at the bottom went through neuronlike morphological changes. These changes were not observed in the absence of activated PBMCs in the insert. (b) When MSCs were incubated for 3 days with the CM from activated PBMCs, they acquired neuronlike morphology. (c) MSCs incubated with the CM from activated PBMCs expressed ChAT, NCAM1, NF-M, and TUJ1.
Fig. 3
Fig. 3
Inflammation induces the expression of NRs and neuron- or NPC-related markers but not glial markers on the MSC surface. (a) RT-PCR was carried out to assess the expression of nestin, Tuj1, MAP2, NF-M, and GFAP in adherent MSCs cocultured with activated PBMCs (24 h). GAPDH served as a loading control. (b) Flow-cytometric analysis of TUJ1, nestin, and GFAP in adherent MSCs cocultured with activated PBMCs (24 h). (c) Immunofluorescence staining of nestin, TUJ1, NCAM1, GFAP, and O4 in MSCs cocultured in the MLR for 48 h. (d) Immunofluorescence staining for nestin, TUJ1, NCAM1, GFAP, and O4 in MSCs cocultured with PHA-activated PBMCs for 48 h. DAPI was used to stain nuclei. (e) RT-PCR analysis of TrkA, TrkB, TrkC, and p75NTR expression in adherent MSCs cocultured with activated PBMCs (24 h). Tuj1 served as a control for an MSC response to inflammation, and GAPDH was employed as a loading control. (f, g) qPCR was conducted to assess the expression of the aforementioned NRs as in (e). (h) Protein expression of TrkA and p75NTR, but not TrkC, was validated by western blotting in the whole MSC extracts (20 μg) after 48 h of coculture. (i, j) TrkA induction in MSCs in response to experimental inflammation was confirmed by immunofluorescence staining. Error bars are indicative of standard deviations.
Fig. 3
Fig. 3
Inflammation induces the expression of NRs and neuron- or NPC-related markers but not glial markers on the MSC surface. (a) RT-PCR was carried out to assess the expression of nestin, Tuj1, MAP2, NF-M, and GFAP in adherent MSCs cocultured with activated PBMCs (24 h). GAPDH served as a loading control. (b) Flow-cytometric analysis of TUJ1, nestin, and GFAP in adherent MSCs cocultured with activated PBMCs (24 h). (c) Immunofluorescence staining of nestin, TUJ1, NCAM1, GFAP, and O4 in MSCs cocultured in the MLR for 48 h. (d) Immunofluorescence staining for nestin, TUJ1, NCAM1, GFAP, and O4 in MSCs cocultured with PHA-activated PBMCs for 48 h. DAPI was used to stain nuclei. (e) RT-PCR analysis of TrkA, TrkB, TrkC, and p75NTR expression in adherent MSCs cocultured with activated PBMCs (24 h). Tuj1 served as a control for an MSC response to inflammation, and GAPDH was employed as a loading control. (f, g) qPCR was conducted to assess the expression of the aforementioned NRs as in (e). (h) Protein expression of TrkA and p75NTR, but not TrkC, was validated by western blotting in the whole MSC extracts (20 μg) after 48 h of coculture. (i, j) TrkA induction in MSCs in response to experimental inflammation was confirmed by immunofluorescence staining. Error bars are indicative of standard deviations.
Fig. 4
Fig. 4
Neurotrophins are induced in PBMCs activated by MLR or PHA. (a) RT-PCR analysis of NGF and BDNF expression in PBMCs after activation by MLR for 24 h. (b) qPCR analysis of NGF and BDNF expression in the samples as described in panel (a). (c) RT-PCR analysis of NGF and BDNF expression in PBMCs after stimulation with PHA for 24 h. (d) qPCR analysis of NGF and BDNF expression in the samples as described in panel (c). (e~h) ELISA quantification of soluble NGF and BDNF in the CM obtained from activated-PBMC cultures (48 h). Three independent experiments were conducted.
Fig. 5
Fig. 5
Inflammatory conditions induce a cholinergic-neuron–like phenotype in MSCs and nAChRs in activated PBMCs. (a) Immunofluorescence staining of ChAT, GABA, and TH in MSCs cocultured with activated PBMCs (48 h). (b) qPCR was carried out to quantify ChAT expression in MSCs after inflammatory stimulation for 24 h. (c) Western blotting confirmed ChAT expression in the whole MSC extract (20 μg) after coculture for 48 h. (d) ACh and choline concentration was measured in the CM obtained from PBMCs alone (P or Po), PHA-activated PBMCs (PPHA), or MLR (P and Po) culture without or with MSCs (n=3). (e) ACh and choline concentration was measured in the CM obtained from PBMCs alone (P or Po) or MLR (P and Po) culture without or with MSCs for 48 h. “MLR sup.” is the supernatant from the MLR without MSCs. “(MLR+MSC) sup.” is the supernatant from MLR with MSCs (n=3). (f) RT-PCR analysis of several nAChR subunits in activated PBMCs. (g) qPCR was carried out to assess nAChR α5 expression in activated PBMCs after incubation for 24 h. (h) qPCR was performed to measure nAChR α7 expression in activated PBMCs after incubation for 24 h. (i) An increase in the protein expression of the nAChR α7 subunit in activated PBMCs was confirmed by western blotting of whole MSC extracts prepared after MLR or PHA stimulation for 48 h.
Fig. 5
Fig. 5
Inflammatory conditions induce a cholinergic-neuron–like phenotype in MSCs and nAChRs in activated PBMCs. (a) Immunofluorescence staining of ChAT, GABA, and TH in MSCs cocultured with activated PBMCs (48 h). (b) qPCR was carried out to quantify ChAT expression in MSCs after inflammatory stimulation for 24 h. (c) Western blotting confirmed ChAT expression in the whole MSC extract (20 μg) after coculture for 48 h. (d) ACh and choline concentration was measured in the CM obtained from PBMCs alone (P or Po), PHA-activated PBMCs (PPHA), or MLR (P and Po) culture without or with MSCs (n=3). (e) ACh and choline concentration was measured in the CM obtained from PBMCs alone (P or Po) or MLR (P and Po) culture without or with MSCs for 48 h. “MLR sup.” is the supernatant from the MLR without MSCs. “(MLR+MSC) sup.” is the supernatant from MLR with MSCs (n=3). (f) RT-PCR analysis of several nAChR subunits in activated PBMCs. (g) qPCR was carried out to assess nAChR α5 expression in activated PBMCs after incubation for 24 h. (h) qPCR was performed to measure nAChR α7 expression in activated PBMCs after incubation for 24 h. (i) An increase in the protein expression of the nAChR α7 subunit in activated PBMCs was confirmed by western blotting of whole MSC extracts prepared after MLR or PHA stimulation for 48 h.
Fig. 6
Fig. 6
MSC-mediated immunosuppression via ACh is reversed by α-BTX (ACh antagonist), and lymphocyte proliferation is inhibited by ACh-Cl (ACh agonist). (a) MSC-suppressed lymphocyte proliferation was significantly restored by α-BTX addition to the MLR medium. (b) MSC-suppressed TNF-α production during MLR was restored by α-BTX treatment. (c) Suppressed secretion of IFN-γ during MLR was significantly restored by α-BTX treatment. (d) MSC-mediated suppression of PHA-stimulated lymphocyte proliferation was significantly attenuated by α-BTX treatment. (e) MSC-suppressed TNF-α production in PHA-activated PBMCs increased in the presence of α-BTX. (f) The reduced IFN-γ secretion by PHA-activated PBMCs was restored by α-BTX. (g, j) ACh-Cl addition to the medium significantly attenuated the increase in lymphocyte proliferation caused by (g) MLR or (j) PHA treatment. (h, k) Production of TNF-α by activated PBMCs elicited by (h) MLR or (k) PHA stimulation was attenuated by ACh-Cl treatment. (i, l) ACh-Cl treatment significantly suppressed the IFN-γ secretion from PBMCs activated by (i) MLR or (l) PHA stimulation. All data are the average of 3~5 independent experiments and were statistically evaluated by paired Student’s t test. p values <0.05 were assumed to indicate statistically significant variations.
Fig. 6
Fig. 6
MSC-mediated immunosuppression via ACh is reversed by α-BTX (ACh antagonist), and lymphocyte proliferation is inhibited by ACh-Cl (ACh agonist). (a) MSC-suppressed lymphocyte proliferation was significantly restored by α-BTX addition to the MLR medium. (b) MSC-suppressed TNF-α production during MLR was restored by α-BTX treatment. (c) Suppressed secretion of IFN-γ during MLR was significantly restored by α-BTX treatment. (d) MSC-mediated suppression of PHA-stimulated lymphocyte proliferation was significantly attenuated by α-BTX treatment. (e) MSC-suppressed TNF-α production in PHA-activated PBMCs increased in the presence of α-BTX. (f) The reduced IFN-γ secretion by PHA-activated PBMCs was restored by α-BTX. (g, j) ACh-Cl addition to the medium significantly attenuated the increase in lymphocyte proliferation caused by (g) MLR or (j) PHA treatment. (h, k) Production of TNF-α by activated PBMCs elicited by (h) MLR or (k) PHA stimulation was attenuated by ACh-Cl treatment. (i, l) ACh-Cl treatment significantly suppressed the IFN-γ secretion from PBMCs activated by (i) MLR or (l) PHA stimulation. All data are the average of 3~5 independent experiments and were statistically evaluated by paired Student’s t test. p values <0.05 were assumed to indicate statistically significant variations.
Fig. 7
Fig. 7
The proposed model of an immunomodulatory mechanism utilized by human MSCs in an inflammatory milieu. The inflammatory conditions drive human MSCs to adopt a neuronlike phenotype. It is probable that the expression of neurotrophins such as NGF and BDNF in human AICs and the presence of NRs on MSCs are associated with these changes in MSCs. Furthermore, the inflammatory milieu probably induces the expression of nAChR including nAChR α7, which participates in the negative regulation of activated lymphocytes. Neuronlike MSCs stimulated by neurotrophins and unknown factors secrete ACh, which binds to AICs via nAChR α7, thereby inhibiting the proliferation and function of AICs.

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