Mouse mesenchymal stem cells inhibit high endothelial cell activation and lymphocyte homing to lymph nodes by releasing TIMP-1

Abstract

Mesenchymal stem cells (MSC) represent a promising therapeutic approach in many diseases in view of their potent immunomodulatory properties, which are only partially understood. Here, we show that the endothelium is a specific and key target of MSC during immunity and inflammation. In mice, MSC inhibit activation and proliferation of endothelial cells in remote inflamed lymph nodes (LNs), affect elongation and arborization of high endothelial venules (HEVs) and inhibit T-cell homing. The proteomic analysis of the MSC secretome identified the tissue inhibitor of metalloproteinase-1 (TIMP-1) as a potential effector molecule responsible for the anti-angiogenic properties of MSC. Both in vitro and in vivo, TIMP-1 activity is responsible for the anti-angiogenic effects of MSC, and increasing TIMP-1 concentrations delivered by an Adeno Associated Virus (AAV) vector recapitulates the effects of MSC transplantation on draining LNs. Thus, this study discovers a new and highly efficient general mechanism through which MSC tune down immunity and inflammation, identifies TIMP-1 as a novel biomarker of MSC-based therapy and opens the gate to new therapeutic approaches of inflammatory diseases.

Figure 1

MSC affect size and cellularity of dLNs. (a) Diagram of the experimental protocol designed to investigate the influence of MSC transplantation. Mice were immunized in the dorsal back with CFA/OVA on day 0 and, on day 1, a group of animals received subcutaneous injection of 10⁶ MSC in the lumbar back. On days 4–5, depending on the subsequent analyses, brachial LNs were collected and processed. (b, c) On day 5, dLNs were digested and analyzed by flow cytometry. Data are representative of eight mice from two independent experiments; (d, e) OPT data are expressed as percentage on OVA average. In (b–e), error bars represent s.e. (*P<0.05; **P<0.01; ***P<0.005; t-test).

Figure 2

MSC inhibit endothelial activation in dLNs. Mice were treated as in Figure 1a and, on day 5, dLNs were collected, stained and analyzed by confocal microscopy. (a, c) 8-μm frozen section was stained with anti-CD31, anti-Lyve-1 and anti-VCAM-1 or anti-ICAM-1, as indicated (10 × , scale bar 200 mm). (b, d) Mander's colocalization coefficient quantifies the degree of overlap. (e) Integrated density quantifies the CD31 and Lyve-1 immunopositivity amount on cross sections of lymph node. In all graphs, error bar represents s.e. (**P<0.01, ***P<0.005; t-test).

Figure 3

MSC inhibit HEV activation and proliferation in vivo. Mice were treated as illustrated in Figure 1a and dLNs were collected, digested and analyzed by flow cytometry. The graphs show (a) the absolute number of CD45^-CD31⁺ cells per single LN expressed as normalized percentage on CFA/OVA (t-test), (b) BrdU incorporation cytometry after 48 h (Mann–Whitney test), (c) HEV cell numbers and (d) mean fluorescence intensity (MFI) of VCAM-1 expression on HEV (t-test) (*P<0.05; **P<0.01).

Figure 4

MSC suppress HEV lengthening and branching. (a–e) Mice were treated as described in Figure 1a, and on day 4 brachial LNs were prepared for OPT imaging (Meca-79 Alexa-594 and B220 Alexa-488). (a) Representative images from OPT scanning (scale bar, 400 μm). (b) Total HEV length per LN. (c) Total HEV volume per LN. (d) Number of HEV segments per LN. (e) Number of branch points per LN. (f, g) 3D immunofluorescence of lymphocyte homing in the presence of MSC tested at day 3 post immunization. (f) Representative images. (g) Absolute counts per mm³ in OVA and OVA+MSC-treated mice, with error bars representing s.e. (*P<0.05, **P<0.01; t-test).

Figure 5

Endothelial cells are a direct target of MSC-secreted molecules. The supernatant of MSC stimulated with IL-1b, IL-6 and TNF-a (MSC-CM) or unstimulated MSC (unst MSC) was collected as described in Materials and methods, and its effect on endothelial cell lines activation was determined. (a, b) SVEC4-10 network formation. Representative images at 6 h and segment length quantification indicated as % of variation in comparison with control condition. Data are expressed as mean±s.e.m. and represent the pool of three experiments (t-test). (c, d) Expression of endothelial adhesion molecules. Representative histograms showing the mean fluorescence intensity (MFI) of VCAM-1 and ICAM-1 on MELC and 1G11 endothelial cell line. (e, f) Quantitative analyses of (c) and (d), respectively (t-test). (g–j) TNF-a induced NF-kB translocation. Representative confocal images ( × 40) of MELC (g) or 1G11 (h) cells stained for NF-kB and phalloidin. Scale bar 10 um. (I, j) Quantification of NF-kB translocation into the nucleus expressed as percentage of the total (one representative experiment out of three; one-way ANOVA) (*P<0.05; **P<0.01; ***P<0.0001).

Figure 6

Distribution into biological processes of the proteins upregulated in MSC-CM. The proteins that were significantly upregulated or present only in MSC-CM were classified into different biological processes according to the GO classification system. (a) The bar chart shows the count of the top 26 most-enriched GO terms in MSC-CM versus unstimulated MSC-CM. Color coding indicates the fold enrichment. (b) Proteins categorized as modulators involved in inflammation processes and/or angiogenesis. The histograms report the GOBP groups related to angiogenesis or inflammation.

Figure 7

TIMP-1 mediates the anti-angiogenic effect of MSC-CM in vitro and the anti-inflammatory effect of MSC in vivo. SVEC4-10 network formation in matrigel in the presence of MSC-CM or unst MSC-CM and anti-TIMP-1 blocking antibody. (a) anti-mTIMP1 blocking antibody restores SVEC4-10 network formation in matrigel in the presence of MSC-CM. Representative images at 6 h (left) and segment length quantification as percentage of variation (right) are shown. Data are expressed as mean±s.e.m. (*P<0.05, **P<0.01; one-way ANOVA). (b) Diagram of the experimental protocol designed to block the TIMP-1 activity during the anti-inflammatory effects of MSC. Mice were immunized in the dorsal region with CFA/OVA on day 0 and, on day 1, three groups of animals received subcutaneous injection of 10⁶ MSC in the lumbar region. Eighteen hours after MSC transplantation, goat polyclonal anti-TIMP-1 IgG or isotype-matched goat IgG was i.v. administrated. On day 4,*-3 brachial LNs were collected, processed and analyzed by flow cytometry; (c, d) the graphs show the absolute number of CD45⁻CD31⁺ cells and HEV PNAd⁺ cells per single LN, expressed as normalized percentage on CFA/OVA (t-test). (e) Diagram of the experimental protocol designed to investigate the contribution of MSC-derived TIMP-1 on dLN endothelium. Mice were immunized in the dorsal region with CFA/OVA on day 0. The day after, two groups of animals received in the lumbar region subcutaneous injection of 10⁶ MSC transfected with either scramble control siRNA or siRNA specific for TIMP-1, respectively. On day 4, brachial LNs were collected, processed and analyzed by flow cytometry; (f, g) graphs showing the absolute number of CD45⁻CD31⁺ cells and HEV PNAd⁺ cells per single dLN. Data are expressed as normalized percentage on CFA/OVA (Mann–Whitney test) (*P<0.05; **P<0.01).

Figure 8

TIMP-1 overexpression in vivo mimics MSC transplantation. (a) Diagram of the experimental protocol designed to overexpress TIMP-1 in immunized mice. One day after AAV9-TIMP-1 or AAV9-LacZ administration (day 0), mice were immunized with CFA/OVA. Brachial dLNs were collected 4 days after immunization and processed for flow cytometry. The graphs show the absolute number of total cells (b), CD45⁺ cells (c), CD45⁻CD31⁺ (d) and HEV PNAd⁺ (e) cells per single LN, expressed as normalized percentage on CFA/OVA. Error bars represent standard error (*P<0.05; **P<0.01; Mann–Whitney test).