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. 2018 Oct 25;9(1):286.
doi: 10.1186/s13287-018-1039-2.

Preconditioning of bone marrow-derived mesenchymal stem cells highly strengthens their potential to promote IL-6-dependent M2b polarization

Denise Philipp  1 , Laura Suhr  1 , Thorsten Wahlers  1 , Yeong-Hoon Choi  1 , Adnana Paunel-Görgülü  2
Affiliations

Preconditioning of bone marrow-derived mesenchymal stem cells highly strengthens their potential to promote IL-6-dependent M2b polarization

Denise Philipp et al. Stem Cell Res Ther. .

Abstract

Background: During the last decade, mesenchymal stem cells (MSCs) have gained much attention in the field of regenerative medicine due to their capacity to differentiate into different cell types and to promote immunosuppressive effects. However, the underlying mechanism of MSC-mediated immunoregulation is not fully understood so far. Macrophages are distinguished in classical activated, pro-inflammatory M1 and alternatively activated M2 cells, which possess different functions and transcriptional profiles with respect to inflammatory responses. As polarization is not fixed, macrophage functional plasticity might be modulated by the microenvironment allowing them to rapidly react to danger signals and maintaining tissue homeostasis.

Methods: Murine MSCs were preconditioned with IL-1ß and IFN-ɣ to enhance their immunosuppressive capacity regarding macrophage polarization under M1- and M2a-polarizing conditions. Macrophage polarization was analyzed by real-time PCR, flow cytometry, and cytokine detection in culture supernatants. The role of MSC-derived nitric oxide (NO), prostaglandin E2 (PGE2), and IL-6 in this process has been evaluated using siRNA transfection and IL-6 receptor-deficient macrophages, respectively.

Results: Preconditioned, but not unprimed, MSCs secreted high levels of NO, IL-6, and PGE2. Co-culture with macrophages (M0) in the presence of M1 inducers (LPS + IFN-ɣ) led to significant reduction of CD86 and iNOS protein in macrophages and diminished TNF-α secretion. Additionally, CD86 and iNOS protein expression as well as NO and IL-10 secretion were markedly increased under M2a-polarizing culture conditions (IL-4). MSC-dependent macrophage polarization did not depend on direct cell-cell contact. Co-culturing in the presence of LPS and IFN-ɣ resulted in the upregulation of M2a, M2b, and M2c marker genes, whereas in the presence of IL-4 only M2b markers were significantly increased. In turn, IL-10-producing regulatory M2b cells significantly inhibited IFN-ɣ expression in CD4+ T lymphocytes. Finally, we show that MSC-mediated macrophage polarization strongly depends on IL-6, whereas a minor role for NO and PGE2 was found.

Conclusions: Preconditioning of MSCs highly strengthens their capacity to regulate macrophage features and to promote immunosuppression. Repression of M1 polarization during inflammation and M2b polarization under anti-inflammatory conditions strongly depend on functional IL-6 signaling in macrophages. The potential benefit of preconditioned MSCs and IL-6 should be considered for future clinical treatment.

Keywords: IL-6; Immunosuppression; Macrophage polarization; Mesenchymal stem cells; Preconditioning.

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Conflict of interest statement

Ethics approval and consent to participate

Cell isolation from mice was approved by the local ethics committee (Bezirksregierung Köln; Germany; No: 4.16.002; 4.16.027).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Figures

Fig. 1
Fig. 1
Characterization of preconditioned MSCs. MSCs were cultured in the presence of 30 ng/ml IFN-ɣ and 3 ng/ml IL-1ß for 24 h. a iNOS gene expression was analyzed by real-time PCR. n = 3, *p < 0.05. b Nitrite (NO2) levels in culture supernatants were quantified by Griess assay. n = 9, ***p < 0.001. c TGF-ß (n = 4), IL-6 (n = 6) and PGE2 (n = 5) levels in culture supernatants were quantified by ELISA. *p < 0.05; ***p < 0.001. IL interleukin, MSCs mesenchymal stem cells, iNOS inducible nitric oxide synthase, PGE2 prostaglandin E2, TGF-ß tumor growth factor beta
Fig. 2
Fig. 2
Preconditioned MSCs impair polarization toward the M1 phenotype. Unstimulated or preconditioned MSCs were co-cultured with macrophages (ratio 1:2) in the presence of M1-polarizing cytokines (20 ng/ml IFN-ɣ + 100 ng/ml LPS) for 24 h. Then, cells were separated using autoMACS. In parallel experiments, MSCs were cultured in transwells (TW) to avoid direct contact to co-cultured macrophages. a The expression of the surface receptors CD86 and CD206 as well as intracellular iNOS expression in macrophages was quantified by flow cytometry. Percentage of positive cells is depicted. For iNOS expression, the mean fluorescence units (MFU) are additionally displayed. n = 7, *p < 0.05, **p < 0.01 vs. macrophages cultured alone (M1); #p < 0.05. b TNF-α levels in culture supernatants were quantified by ELISA and NO2 levels were quantified by Griess assay. Results are representative for seven independent experiments. *p < 0.05, **p < 0.01 vs. macrophages cultured alone (M1); #p < 0.05. M macrophage, MSCs mesenchymal stem cells, iNOS inducible nitric oxide synthase, NO2 nitrite, TNF-α tumor necrosis factor alpha
Fig. 3
Fig. 3
Preconditioned MSCs drive M2 polarization in the presence of IL-4. Unstimulated or preconditioned MSCs were co-cultured with macrophages (ratio 1:2) in the presence of 20 ng/ml IL-4 for 24 h. After cell separation by autoMACS, macrophages were used for flow cytometric analyses. In some experiments, MSCs were placed in transwell chambers (TW). a The expression of the surface markers CD86 and CD206 as well as intracellular iNOS expression in macrophages was quantified by flow cytometry. Percentage of positive cells is depicted. For iNOS expression, the mean fluorescence intensity is additionally displayed. n = 7, *p < 0.05 vs. macrophages cultured alone (M2); #p < 0.05. b Levels of IL-10 in cell culture supernatants were measured by ELISA. NO2 concentrations were quantified by Griess assay. Results are representative for five independent experiments. *p < 0.05 vs. macrophage control (M2); #p < 0.05, &p < 0.01. IL interleukin, M macrophage, MSCs mesenchymal stem cells, iNOS inducible nitric oxide synthase, NO2 nitrite
Fig. 4
Fig. 4
Preconditioned MSCs promote M2 polarization of M1-like cells and M2b polarization of M2a-like cells. MSCs and preconditioned MSCs (acMSCs) were placed in transwells to avoid direct cell interactions and were further co-cultured with differentiated macrophages (M0) under M1- (a) and M2- (b) polarizing culture conditions for 24 h (ratio 1:2). Marker genes specific for M2a (Ym-1), M2b (SPHK1, LIGHT), and M2c (MertK) were analyzed by real-time PCR. In addition, the expression of pro-inflammatory IL-6 and TNF-α were investigated. n = 8–10, *p < 0.05, **p < 0.01, ***p < 0.001 vs. control macrophages (M1 and M2). #p < 0.05, ##p < 0.01, ###p < 0.001. FIZZ1/RELMα: found in inflammatory zone 1/resistin-like molecule alpha, IL interleukin, LIGHT lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, M macrophage, MertK tyrosine-protein kinase MER, MSCs mesenchymal stem cells, acMSCs activated, preconditioned mesenchymal stem cells, SPHK1, sphingosine kinase 1, TNF-α tumor necrosis factor alpha, Ym-1 chitinase-like protein
Fig. 5
Fig. 5
MSC-dependent modulation of macrophage plasticity in IL6Rα-deficient cells. Preconditioned MSCs (acMSCs) were co-cultured with wild type macrophages or macrophages isolated from the bone marrow of IL-6Rα-deficient (IL6R-KO) mice. For differentiation toward an M1-like and M2a-like phenotype, cells were treated with 20 ng/ml IFN-ɣ + 100 ng/ml LPS (a) or 20 ng/ml IL-4 (b), respectively, for 24 h. Gene expression of IL-6, TNF-α, M2a markers (Ym-1, FIZZ1/RELMα), M2b markers (SPHK1, LIGHT), and the M2c marker MertK in macrophages was analyzed by real-time PCR. Results are representative for six to nine independent experiments and are expressed as fold change vs. expression found in macrophages. *p < 0.05, **p < 0.01 vs. gene expression found in wild type macrophages (M1 or M2) co-cultured with preconditioned MSCs (acMSCs). FIZZ1/RELMα: found in inflammatory zone 1/resistin-like molecule alpha, IL interleukin, IL-6Rα interleukin 6 receptor alpha, KO knockout, LIGHT lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, M macrophage, MertK tyrosine-protein kinase MER, MSCs mesenchymal stem cells, acMSCs activated, preconditioned mesenchymal stem cells, SPHK1, sphingosine kinase 1, TNF-α tumor necrosis factor alpha, Ym-1 chitinase-like protein
Fig. 6
Fig. 6
Role of IL-6 for macrophage polarization toward M2b phenotype. a Preconditioned MSCs (acMSCs) were co-cultured with wild type macrophages or macrophages isolated from IL-6Rα-deficient (IL6R-KO) mice in the presence of IL-4 (20 ng/ml). After 18 h, the expression of pSTAT3 (Tyr705) was verified by Western blot. STAT3 served as loading control. One representative Western blot of three independent experiments is depicted. b Wild type macrophages were treated with 20 ng/ml IL-4 and 25 ng/ml IL-6 for 24 h. Then, the expression of macrophage-specific markers and TNF-α was analyzed by real-time PCR. n = 6, *p < 0.05. FIZZ1/RELMα, found in inflammatory zone 1/resistin-like molecule alpha, IL interleukin, IL-6Rα interleukin 6 receptor alpha, KO knockout, LIGHT lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, M macrophage, MertK tyrosine-protein kinase MER, MSCs mesenchymal stem cells, acMSCs activated, preconditioned mesenchymal stem cells, SPHK1 sphingosine kinase 1, STAT3 Signal transducer and activator of transcription 3, pSTAT3 phosphorylated STAT3, TNF-α tumor necrosis factor alpha, WT wild type, Ym-1 chitinase-like protein
Fig. 7
Fig. 7
NO and PGE2 are only partially involved in MSC-mediated macrophage polarization. MSCs were transfected with iNOS- or COX-2-specific siRNAs to reduce NO and PGE2 secretion and further stimulated with IFN-ɣ and IL-1ß. Transfected, activated MSCs (acMSCs+siRNA) were co-cultured with M0 macrophages under M1- (20 ng/ml IFN-gamma + 100 ng/ml LPS, a) and M2a- (20 ng/ml IL-4, b) polarizing culture conditions for 24 h. Preconditioned MSCs (acMSCs) served as controls. Gene expression of IL-6, TNF-α, M2a markers (Ym-1, FIZZ1/RELMα), M2b markers (SPHK1, LIGHT), and the M2c marker MertK in macrophages was analyzed by real-time PCR. Results are representative for six to eight independent experiments and are expressed as fold change vs. expression found in macrophages. *p < 0.05, **p < 0.01. FIZZ1/RELMα, found in inflammatory zone 1/resistin-like molecule alpha, IL interleukin, LIGHT lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, M macrophage, MertK tyrosine-protein kinase MER, MSCs mesenchymal stem cells, acMSCs activated, preconditioned mesenchymal stem cells, SPHK1, sphingosine kinase 1, TNF-α tumor necrosis factor alpha, Ym-1 chitinase-like protein
Fig. 8
Fig. 8
M2b-polarized macrophages produce high levels of IL-10 and impair IFN-ɣ expression in CD4+ naïve T lymphocytes. a IL-10 gene expression was proven in macrophages isolated from wild type macrophages (M2) after co-culture with preconditioned MSCs and IL-4 or treatment with recombinant IL-4 (20 mg/ml) + IL-6 (25 ng/ml), respectively. Expression of IL-10 in cells from IL-6Rα-deficient mice (IL6R-KO M2) after co-culture with preconditioned MSCs + IL-4 was additionally analyzed. Fold change in gene expression vs. control cells is depicted. n = 6, *p < 0.05; ***p < 0.001. b Wild type macrophages pre-cultured with or without preconditioned MSCs and IL-4 for 24 h were further co-cultured with CD4+ naïve T lymphocytes allowing direct cell-cell interactions for further 24 h. Gene expression of Th1 markers GATA-3 and IFN-ɣ as well as IL-2 and IL-4 (Th2) was analyzed by real-time PCR. n = 5, *p < 0.05. IL interleukin, IFN-ɣ interferon gamma, M macrophage, T lymphocyte
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References

    1. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143–147. doi: 10.1126/science.284.5411.143. - DOI - PubMed
    1. Tropel P, Platet N, Platel JC, Noel D, Albrieux M, Benabid AL, Berger F. Functional neuronal differentiation of bone marrow-derived mesenchymal stem cells. Stem Cells. 2006;24(12):2868–2876. doi: 10.1634/stemcells.2005-0636. - DOI - PubMed
    1. Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, Mavilio F. Muscle regeneration by bone marrow-derived myogenic progenitors. Science. 1998;279(5356):1528–1530. doi: 10.1126/science.279.5356.1528. - DOI - PubMed
    1. English K. Mechanisms of mesenchymal stromal cell immunomodulation. Immunol Cell Biol. 2013;91(1):19–26. doi: 10.1038/icb.2012.56. - DOI - PubMed
    1. Crisostomo PR, Wang Y, Markel TA, Wang M, Lahm T, Meldrum DR. Human mesenchymal stem cells stimulated by TNF-alpha, LPS, or hypoxia produce growth factors by an NF kappa B- but not JNK-dependent mechanism. Am J Physiol Cell Physiol. 2008;294(3):C675–C682. doi: 10.1152/ajpcell.00437.2007. - DOI - PubMed

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