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Differential TLR activation of murine mesenchymal stem cells generates distinct immunomodulatory effects in EAE - PubMed

  • ️Fri Jan 01 2016

Differential TLR activation of murine mesenchymal stem cells generates distinct immunomodulatory effects in EAE

Ana María Vega-Letter et al. Stem Cell Res Ther. 2016.

Abstract

Background: Recently, it has been observed that mesenchymal stem cells (MSCs) can modulate their immunoregulatory properties depending on the specific in-vitro activation of different Toll-like receptors (TLR), such as TLR3 and TLR4. In the present study, we evaluated the effect of polyinosinic:polycytidylic acid (poly(I:C)) and lipopolysaccharide (LPS) pretreatment on the immunological capacity of MSCs in vitro and in vivo.

Methods: C57BL/6 bone marrow-derived MSCs were pretreated with poly(I:C) and LPS for 1 hour and their immunomodulatory capacity was evaluated. T-cell proliferation and their effect on Th1, Th17, and Treg differentiation/activation were measured. Next, we evaluated the therapeutic effect of MSCs in an experimental autoimmune encephalomyelitis (EAE) model, which was induced for 27 days with MOG35-55 peptide following the standard protocol. Mice were subjected to a single intraperitoneal injection (2 × 106 MSCs/100 μl) on day 4. Clinical score and body weight were monitored daily by blinded analysis. At day 27, mice were euthanized and draining lymph nodes were extracted for Th1, Th17, and Treg detection by flow cytometry.

Results: Pretreatment of MSCs with poly(I:C) significantly reduced the proliferation of CD3+ T cells as well as nitric oxide secretion, an important immunosuppressive factor. Furthermore, MSCs treated with poly(I:C) reduced the differentiation/activation of proinflammatory lymphocytes, Th1 and Th17. In contrast, MSCs pretreated with LPS increased CD3+ T-cell proliferation, and induced Th1 and Th17 cells, as well as the levels of proinflammatory cytokine IL-6. Finally, we observed that intraperitoneal administration of MSCs pretreated with poly(I:C) significantly reduced the severity of EAE as well as the percentages of Th1 and Th17 proinflammatory subsets, while the pretreatment of MSCs with LPS completely reversed the therapeutic immunosuppressive effect of MSCs.

Conclusions: Taken together, these data show that pretreatment of MSCs with poly(I:C) improved their immunosuppressive abilities. This may provide an opportunity to better define strategies for cell-based therapies to autoimmune diseases.

Keywords: Autoimmunity; Experimental autoimmune encephalomyelitis; Immunomodulation; Mesenchymal stem cells; Toll-like receptors 3 and 4.

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Figures

Fig. 1
Fig. 1

Characterization of murine MSCs and TLR expression. (a) Morphology and characteristics of murine BM-MSCs (20× magnification). (b) Immunophenotypic profile from a representative murine MSC population. Cell surface markers, solid red; isotype cell controls, gray. (c) MSCs were confirmed to have the capacity to differentiate into chondrocytes, osteoblasts, and adipocytes (right) by alizarin red, safranin O, and oil red staining (4, 10, and 40× magnification, respectively), as described in Methods. Respective controls (culture medium only, without differentiation conditions) (Left). (d) Relative expression of TLR3 and TLR4 in MSCs confirmed by RT-qPCR normalized with 18S = 1 × 10–5 as described previously [37] (n = 3). (e) RT-qPCR products were analyzed by 2.5 % agarose gel electrophoresis along with a low-range DNA ladder (MW). Negative control, without cDNA template. MW molecular weight. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2
Fig. 2

Immunomodulatory capacity of MSCs stimulated with TLR3 and TLR4 ligands in vitro. (a) To study the immunosuppressive capacity of untreated MSCs and MSCs pretreated for 1 hour with poly(I:C) or LPS on T-cell response, we performed an in-vitro T-cell stimulation assay at different ratios of MSCs:splenocytes, as described in Methods. MSCs were either unstimulated or were stimulated with poly(I:C) (10 μg/ml) or LPS (500 ng/ml) for 1 hour before being cocultured with T cells in complete RPMI medium. Previously, splenocytes were labeled with CTV and stimulated with Con A and finally cultured with MSCs at 1:5, 1:10, or 1:20 ratios for 3 days. T-cell proliferation was evaluated by flow cytometry, gating on CD3+ cells. (b) Secretion of nitric oxide (NO) by MSCs in coculture with splenocytes at a 1:10 ratio was measured using a modified Griess reagent. (c) IL-6 mRNA expression evaluated by RT-qPCR. (d) IL-6 secretion, measured by ELISA. Data expressed as the mean ± SED. A Mann–Whitney test was performed, *p < 0.05, **p < 0.01,***p < 0.001. MSCsPoly MSCs pretreated with poly(I:C) for 1 hour, MSCsLPS MSCs pretreated with LPS for 1 hour

Fig. 3
Fig. 3

MSCs stimulated with TLR3 and TLR4 ligands differentially modulate Th1 and Th17 differentiation and proliferation. T-helper cell differentiation (a, b, e, f) and proliferation (c, d, g, h) were assessed using naïve CD4+ T cells. Purified CD4+ cells were stimulated with a specific cocktail of cytokines, as described in Methods, to induce Th1 (a–d) and Th17 (e–h) differentiation in the absence or presence of MSCs pretreated with or without a TLR agonist. MSCs were either unstimulated or were stimulated with poly(I:C) (10 μg/ml) or LPS (500 ng/ml) for 1 hour before being cocultured with CD4+ T cells in complete RPMI medium. MSCs were added at day 0 of the differentiation process in a 1:10 ratio (MSCs:T cells). Flow cytometry analysis, gating on CD4+ cells, and intracellular staining, using antibodies (mAb) for IFN-γ and IL17 to identify Th1 and Th17 lymphocytes, respectively, were performed. Representative density plots of six different experiments for Th1 and Th17 differentiation are shown. For proliferation analysis, CD4+ cells were previously labeled with CellTrace Violet (CTV) and analyzed (presented as histograms). Further analysis of the events of each cycle, described by the proliferation index (d, h). Th1 differentiation (b) and proliferation (d) with the MSCs pretreated with poly(I:C) and LPS. Th17 differentiation (f) and proliferation (h) with the MSCs pretreated with poly(I:C) and LPS. Bars represent the mean ± SEM, significant differences calculated using the Mann–Whitney test. *p < 0.05, **p < 0.001. MSCsPoly MSCs pretreated with poly(I:C) for 1 hour, MSCsLPS MSCs pretreated with LPS for 1 hour

Fig. 4
Fig. 4

MSCs treated with poly(I:C) or LPS generate distinct immunomodulatory effects in an EAE model. Data show EAE clinical signs according to the different treatments of MSCs (either pretreated or not with TLR3 and TLR4 ligands). EAE was induced in C57BL/6 mice (n = 10/group) by subcutaneous immunization with 50 μg of MOG35–55 peptide, as described in Methods. MSC controls and MSCs pretreated with poly(I:C) or LPS were injected i.p. at day 4 (2 × 106/mouse) as described in Methods. (a) Scores were measured daily at the same time for 27 days and given a value of 0–5 according to loss of mobility in the lower and upper extremities. (b) The sum of the scores from day 10 to the end of the experiment was pooled by treatment group. Poly(I:C)-pretreated MSCs show a significantly lower cumulative score than that of EAE control mice. (c) Body weight loss (%), measured daily and clustered by treatment. A Wilcoxon rank test was used for comparisons with the untreated MSCs. *EAE + MSCsPoly compared with EAE and **EAE + MSCsLPS compared with EAE + MSCs with the matched pairs test, p < 0.001. MSCsPoly MSCs pretreated with poly(I:C) for 1 hour, MSCsLPS MSCs pretreated with LPS for 1 hour

Fig. 5
Fig. 5

Pretreatment of MSCs with poly(I:C) and LPS generated a differential modulation of Th1 and Th17 cells in EAE mice. Lymph nodes were removed from different groups of treatments at day 27. (a) Th1 detection using CD4+IFN-γ for the five groups of mice. (b) Th17 detection (CD4+IL-17+) for the five groups of mice. Bars represent the mean ± SEM, significant differences calculated using t tests (*p < 0.05, **p < 0.001). MSCsPoly MSCs pretreated with poly(I:C) for 1 hour, MSCsLPS MSCs pretreated with LPS for 1 hour

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