Sphingosine-1-phosphate receptor antagonism enhances proliferation and migration of engrafted neural progenitor cells in a model of viral-induced demyelination

Abstract

The oral drug FTY720 affects sphingosine-1-phosphate (S1P) signaling on targeted cells that bear the S1P receptors S1P1, S1P3, S1P4, and S1P5. We examined the effect of FTY720 treatment on the biology of mouse neural progenitor cells (NPCs) after transplantation in a viral model of demyelination. Intracerebral infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in an acute encephalomyelitis, followed by demyelination similar in pathology to the human demyelinating disease, multiple sclerosis. We have previously reported that intraspinal transplantation of mouse NPCs into JHMV-infected animals resulted in selective colonization of demyelinated lesions, preferential differentiation into oligodendroglia accompanied by axonal preservation, and increased remyelination. Cultured NPCs expressed transcripts for S1P receptors S1P1, S1P2, S1P3, S1P4, and S1P5. FTY720 treatment of cultured NPCs resulted in increased mitogen-activated protein kinase phosphorylation and migration after exposure to the chemokine CXCL12. Administration of FTY720 to JHMV-infected mice resulted in enhanced migration and increased proliferation of transplanted NPCs after spinal cord engraftment. FTY720 treatment did not improve clinical disease, diminish neuroinflammation or the severity of demyelination, nor increase remyelination. These findings argue that FTY720 treatment selectively increases NPC proliferation and migration but does not either improve clinical outcome or enhance remyelination after transplantation into animals in which immune-mediated demyelination is initiated by the viral infection of the central nervous system.

Figure 1

FTY720 treatment activates cultured neural progenitor cells (NPCs). Neurospheres were isolated from the subventricular zone of sphingosine-1-phosphate receptor 1 (S1P1) enhanced green fluorescent protein (eGFP) neonatal pups. A: Representative immunofluorescence images confirm that neurospheres express S1P1, as evidenced by GFP expression. B: Analysis of S1P receptor expression by NPCs at the mRNA level demonstrates expression of transcripts specific for S1P1 to S1P5; the sequence of amplicons confirmed primer specificity. C: Western blot analysis of cultured NPCs treated with either vehicle or 100 nmol/L phosphorylated active form of FTY720 (FTY720P) reveals increased phosphorylation over time. D: Quantitative analysis of Western blot data confirms increased phosphorylation of mitogen-activated protein kinase (MAPK). Analyses of band intensity on films are presented as the relative ratio of phosphorylated MAPK/actin. BF, brightfield microscopy; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2

FTY720 does not affect neural progenitor cell (NPC) differentiation. Exposure of cultured NPCs to daily 100 nmol/L phosphorylated active form of FTY720 (FTY720P) for 5 days does not influence lineage fate commitment to either oligodendroglia (Olig2; A), neurons [mitogen-activated protein (Map) 2; B], and astrocytes [glial fibrillary acidic protein (GFAP); C] compared with vehicle-control–treated cultures. D: Quantification of immunocytochemical staining for defined cell lineages indicates similar frequencies of Olig2-, MAP2-, and GFAP-positive cells after treatment of cultured NPCs with either vehicle or FTY720P. Data were presented as means ± SEM (D). n = 3 independent experiments (D).

Figure 3

FTY720 treatment enhances migration of engrafted green fluorescent protein (GFP)–neural progenitor cells (NPCs). JHMV-infected mice were treated with FTY720 (3 mg/kg daily via i.p. injection) or vehicle control beginning at day 13 postinfection (p.i.). GFP-expressing NPCs were transplanted into the spinal cords at day 14 p.i., and migration of transplanted cells rostral and caudal to the implantation site was assessed 3 weeks posttransplant (p.t.). A: Transplanted GFP-NPCs migrate both rostral and caudal from the implantation site in both control and FTY720-treated mice. Images represent spinal cord sections rostral (1 to 4) and caudal (5 to 8) from the transplantation site. B: Transplanted GFP-NPCs congregate within areas of demyelination located in the anterior and lateral funiculus in both FTY720-treated mice and vehicle control. C: Quantification of GFP-NPC cell numbers at defined spinal cord sections rostral and caudal to the implantation site in vehicle control and FTY720-treated animals. D: Representative images depicting Ki-67 staining by transplanted GFP-NPCs in vehicle control and FTY720-treated mice. Arrowheads represent Ki-67⁺ transplanted GFP-NPCs. E: Quantification of GFP-NPCs expressing Ki-67. Data are presented as means ± SEM (C and E). n = 2 or more independent experiments with n = 4 or more mice per group (C and E). ^∗P < 0.05, ^∗∗P < 0.01.

Figure 4

FTY720 does not affect CXCR4 expression by neural progenitor cells (NPCs). A: Cultured NPCs were treated with increasing concentrations of phosphorylated active form of FTY720 (FTY720P; 10 nmol/L, 100 nmol/L, and 1 μmol/L), and migration in response to 200 ng/mL recombinant mouse CXCL12 was determined after 16 hours. Exposure to FTY720P results in a dose-dependent migration response. B: Representative flow analysis reveals that FTY720 treatment does not affect surface expression of CXCR4 by cultured NPCs at a defined concentration. C: This is confirmed by quantification of mean fluorescence intensity of CXCR4 expression of cultured cells under experimental conditions. Data are given as means ± SEM (A and C). n = 2 independent experiments (A); n = 3 independent experiments (C). ^∗∗∗P < 0.001. Max, maximum.

Figure 5

FTY720 does not decrease the severity of demyelination. Mice were infected with 150 plaque-forming units of JHMV and 3 mg/kg FTY720 or control vehicle treatment initiated at day 13 postinfection (p.i.) and transplanted with green fluorescent protein (GFP)–neural progenitor cells (NPCs) at day 14 p.i. In addition, JHMV-infected mice treated with FTY720 or vehicle alone served as an additional control. A: Representative glutathione S-transferase (GST)-π immunofluorescence staining of spinal cords isolated at day 14 posttransplant (p.t.) from JHMV-infected mice engrafted with GFP-NPCs at day 14 p.i. and treated with either FTY720 or control at day 13 p.i. Arrowheads represent GST-π–positive transplanted GFP-NPCs. B: Similar frequencies of GFP-positive mature oligodendrocytes in GFP-NPC–transplanted mice treated with either FTY720 or vehicle. Twelve spinal cord sections per mouse were counted to determine the frequency of transplanted GFP-NPCs that differentiated into GST-π–positive cells. C: Representative luxol fast blue–stained spinal cord sections from NPC-transplanted mice treated with either FTY720 or control vehicle, or nontransplanted mice treated with FTY720 or control vehicle at day 14 p.t. D: Quantification of demyelination indicates no differences in the severity of white matter damage in experimental groups of mice at day 14 p.t. Data are presented as means ± SEM (B and D). n = 2 independent experiments with n = 4 or more mice per experimental group (B); n = 2 independent experiments with n = 5 or more mice per experimental group (D). Scale bar = 50 μm (A).

Figure 6

FTY720 treatment does not promote remyelination. A: Representative transverse spinal cord section; boxed areas indicate the regions in which demyelinated and remyelinated axons were determined. Representative electron microscopic (EM) images of spinal cords of JHMV–infected mice treated with vehicle (VEH) alone (B), FTY720 (C), green fluorescent protein (GFP)–neural progenitor cells (NPCs) and vehicle (D), and GFP-NPCs and FTY720 (E). Black arrows indicate myelinated axons; white arrows, demyelinated axons; asterisks, remyelinated axons. F: Calculation of g-ratio, as a measurement of axonal remyelination, shows no significant differences between experimental mice. Data are presented as means ± SEM (F). n = 3 per group with n = 150 or more axons per mouse analyzed (F). Original magnification, ×1200 (B–E).

Figure 7

Treatment with FTY720 does not affect neuroinflammation in JHMV infected mice. The effect of FTY720 treatment on T-cell infiltration into the spinal cord after either JHMV infection alone or GFP-NPCs transplantation was examined. Spinal cords were removed at day 28 postinfection [p.i.; day 14 posttransplant (p.t.) of GFP-NPCs], and the frequency of total T-cell subsets and virus-specific T cells was determined. There is no difference in frequencies of CD4⁺ (A) or CD8⁺ (B) T cells and virus-specific CD4⁺ (A) or CD8⁺ (B) T cells in JHMV-infected mice treated with either vehicle control or FTY720. FTY720 does not reduce overall frequencies of infiltrating CD4⁺ (C) or CD8⁺ (D) T cells compared with vehicle-treated control mice nor are there differences in the frequencies of virus-specific CD4⁺ (C) and CD8⁺ (D) T cells following spinal cord transplantation of GFP-NPCs. Data represent means ± SEM (A–D). n = 4 mice or more per group (A–D).

Figure 8

FTY720 induces lymphopenia and down-regulates sphingosine-1-phosphate receptor 1 (S1P1) on T cells. Frequencies of CD4⁺ and CD8⁺ T cells in the blood day 7 posttransplant with GFP-NPCs into JHMV-infected S1P1 eGFP mice treated daily with FTY720 or control starting at day 13 postinfection. FTY720 significantly diminishes the frequency of both CD4⁺ (A) and CD8⁺ (C) T cells and S1P1 expression measured by GFP expression on CD4⁺ (B) and CD8⁺ (D) T cells. Data are presented as means ± SEM (A–D). n = 2 or more experiments with a minimum n = 4 per group (A–D). ^∗P < 0.05. FSC, forward scatter.