Tissue plasminogen activator worsens experimental autoimmune encephalomyelitis by complementary actions on lymphoid and myeloid cell responses

Abstract

Background: Tissue plasminogen activator (tPA) is a serine protease involved in fibrinolysis. It is released by endothelial cells, but also expressed by neurons and glial cells in the central nervous system (CNS). Interestingly, this enzyme also contributes to pathological processes in the CNS such as neuroinflammation by activating microglia and increasing blood-brain barrier permeability. Nevertheless, its role in the control of adaptive and innate immune response remains poorly understood.

Methods: tPA effects on myeloid and lymphoid cell response were studied in vivo in the mouse model of multiple sclerosis experimental autoimmune encephalomyelitis and in vitro in splenocytes.

Results: tPA^-/- animals exhibited less severe experimental autoimmune encephalomyelitis than their wild-type counterparts. This was accompanied by a reduction in both lymphoid and myeloid cell populations in the spinal cord parenchyma. In parallel, tPA increased T cell activation and proliferation, as well as cytokine production by a protease-dependent mechanism and via plasmin generation. In addition, tPA directly raised the expression of MHC-II and the co-stimulatory molecules CD80 and CD86 at the surface of dendritic cells and macrophages by a direct action dependent of the activation of epidermal growth factor receptor.

Conclusions: Our study provides new insights into the mechanisms responsible for the harmful functions of tPA in multiple sclerosis and its animal models: tPA promotes the proliferation and activation of both lymphoid and myeloid populations by distinct, though complementary, mechanisms.

Keywords: Antigen-presenting cells; Experimental autoimmune encephalomyelitis; Neuroinflammation; T cell response; Tissue plasminogen activator.

Fig. 1

tPA^-/- mice show less EAE symptoms than WT mice. a Mean EAE clinical score, b incidence, c peak score, d cumulative clinical score, and e severity index for WT and tPA^-/- mice. Results are expressed as mean + SEM. N = 29 (WT) and N = 35 (tPA^-/-). ^*P < 0.05 and ^**P < 0.01

Fig. 2

tPA^-/- mice show reduced CD4⁺ T cell infiltration than WT in the spinal cord. Absolute number of infiltrated a CD4⁺, b CD8⁺, and c FoxP3⁺ T cells in spinal cords of WT and tPA^-/- EAE mice measured by Malassez cell counting and flow cytometry. Results are expressed as mean + SEM. N = 6 (WT) and N = 3 (tPA^-/-). ^*P < 0.05. d Percentages of T cell subtypes relative to total CD3⁺ T cell number in the spinal cord of tPA^-/- and WT EAE mice. e Photomicrographs show representative immunofluorescence staining (from N = 3 for tPA^-/-and WT) for CD3, CD4, and COLIV markers in the lower thoracic region of mice spinal cords. Nuclei were counterstained with DAPI (blue). f Scheme describing the different parts of the spinal cord. g and h Quantification of the average density of CD4⁺ T cells within infiltrated areas in g the whole spinal cord and in h individual segments (Cerv. cervical, Upper Th. upper thoracic, Lower Th. lower thoracic, L/Sacr. lumbar/sacral. (N = 3). ^*P < 0.05. i and j Intracytoplasmic cytokine detection by flow cytometry in splenocytes from tPA^-/- and WT EAE mice (10 days post-immunization) after stimulation with PMA/ionomycin. Graphs show the percentage of CD4⁺/IFN-γ⁺ T cells and CD4⁺/IL-17⁺ T cells. N = 7 (WT) and N = 7 (tPA^-/-). ^**P < 0.01

Fig. 3

Endogenous tPA stimulates CD4⁺ T cell proliferation and activation. tPA^-/- and WT splenocytes were activated with anti-CD3 and anti-CD28 antibodies (both 1 μg/mL) and treated in the indicated conditions for 4 days. a Representative flow cytometry histograms for proliferation (CFSE) and activation (CD25⁺) of T cells. Corresponding quantification of proliferation index for b CD4⁺and c CD8⁺ T cells. Percentage of increase of CD25 for d CD4⁺ and e CD8⁺ T cells. Results are expressed as mean + SEM (N = 5). ^*P < 0.05

Fig. 4

tPA activates CD4⁺ and CD8⁺, but not FoxP3⁺ T cells in vitro by a proteolytic mechanism. Splenocytes from naive mice were activated with anti-CD3 and anti-CD28 antibodies (both 1 μg/m) and treated in the indicated conditions for 4 days. a Proliferation estimated by observation under bright field binocular (left) and representative flow cytometry histograms for CFSE fluorescence (right), indicating the number of cells in proliferation state at the time of the experiment. Quantification of proliferation index (CFSE) and CD25⁺ MFI (as index of activation) for b, c CD4⁺ and d, e CD8⁺ (N = 4–9). f Quantification of proliferation index for FoxP3⁺ T cells (N = 3). Proliferation index are expressed as mean + SEM percentage vs control (Ctrl = 100% baseline). Activation is expressed as mean + SEM percentage of increase of CD25 MFI. g Quantification of CD4⁺/CCR6⁺ T and CD4⁺/CXCR3⁺ T cells (N = 4). b–g ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs control. h–j Proliferation index of CD4⁺ and CD8⁺ T cells in the indicated treatment conditions. N = 5–9; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs control; ^#P < 0.05 vs tPA. k, l Cytokine measurements (percentage of control) in activated splenocytes treated in the indicated conditions (N = 4–7). ^*P < 0.05, ^**P < 0.01 vs control; ^#P < 0.05 vs tPA

Fig. 5

tPA^-/- mice show reduced myeloid cell infiltration than WT in the spinal cord. Absolute number of infiltrated a DCs and b Mɸ⁺ activated microglia in half-spinal cords of WT and KO EAE mice measured by Malassez cell counting and flow cytometry. Results are expressed as mean + SEM [N = 6 (WT) and N = 3 (tPA^-/-)]. ^*P < 0.05

Fig. 6

tPA polarizes splenic APCs from EAE mice towards a pro-inflammatory phenotype. Splenocytes extracted from EAE mice at the peak of the clinical course were treated in the indicated conditions. a, b Percentage of a CD11c⁺MHC-II⁺-DCs and b F4/80⁺ MHC-II⁺-Mɸ in each APC subpopulation. c, d MFI of MHC-II in APCs, e–h Percentage of e, f MHC-II⁺ CD80⁺ CD86⁺ immunogenic and g, h and MHC-II⁺ CD80^- CD86^- tolerogenic APCs in the presence of different tPA concentrations. In all cases, tPA at the concentrations of 0.2 and 20 μg/mL showed no differences. Results are expressed as mean + SEM (N = 5), ^*P < 0.05

Fig. 7

tPA stimulates MOG_35–55-dependent APC maturation and T cell proliferation via its EGF-like domain. Splenocytes extracted from EAE mice at the peak of the clinical course were treated in the indicated conditions. a, b MHC-II MFI in a CD11c⁺MHC-II⁺-DCs and b F4/80⁺ MHC-II⁺-MΦs exposed to 2 μg/mL of tPA or 2 μg/mL of tPA-GGACK. c, d Expression of MHC-II MFI in c CD11c⁺MHC-II⁺-DCs and d F4/80⁺ MHC-II⁺-MΦs after combined treatment with 2 μg/mL of tPA and 5-μM EGFR inhibitor AG1478. e, f Cell proliferation index of e CD4⁺ and f CD8⁺ T cells after combined treatment with 5 μg/mL MOG_35–55, 2 μg/mL of tPA and 5-μM EGFR inhibitor AG1478. N = 6–7 and ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs control; ^#P < 0.05, ^###P < 0.001 vs tPA. g, h Cell proliferation index of sorted g CD4⁺ and h CD8⁺ T cells in co-culture with MOG-stimulated CD3 T cell-depleted splenocytes with/without 2 μg/mL of tPA during 24 or 96 h (N = 6–7 for a–f, N = 3 for g and h, and ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs baseline; ^#P < 0.05, ^###P < 0.001 vs tPA)