Bone Marrow Transfer in Relapsing-Remitting EAE Ameliorates Disease at First Remission, with No Synergistic Effect upon Co-Transplantation with Mesenchymal Stem Cells

Abstract

Multiple sclerosis (MS) is a neurological disorder characterized by an autoimmune response, demyelinating plaques and axonal damage. Intense immunosuppression (II) followed by autologous hematopoietic stem cell transplantation has been proposed as a treatment in severe forms of MS. We have used murine relapsing-remitting (RR) experimental autoimmune encephalomyelitis (RR-EAE) to evaluate the transplantation of syngeneic bone marrow cells (BMC) after II, in combination with mesenchymal stem cells (MSCs) as a new therapeutic adjunct capable of improving immune reconstitution. In EAE-affected mice treated with BMC alone, we observed a drastic reduction in the clinical course only during the early RR phase of the disease. There was no difference in the RR-EAE clinical course between mice treated with BMC alone and co-transplanted mice. To analyze the immune reconstitution, we quantified the circulating immune cells in naïve and RR-EAE-affected mice after II, with BMC alone or in combination with MSC. Although II resulted in reduced numbers of circulating immune cells, reconstitution did not differ in co-transplanted mice. During the early phase of the disease, IL-4 was significantly elevated in co-transplanted mice, as compared to those treated with BMC alone. These data suggest that BMC transplantation after II transiently ameliorates the clinical symptoms of RR-EAE, but that co-transplantation with MSC has no synergistic effect.

Keywords: bone-marrow cells; experimental autoimmune encephalomyelitis; intensive immunosuppression; mesenchymal stem cells; multiple sclerosis; stem cell therapy.

Figure 1

Mice treated with HSCs after intense immunosuppression develop milder EAE than their untreated counterparts only during the first relapsing-remitting phase. (A) EAE course of mice untreated or irradiated and treated with BMC. A representative example of two independent experiments is presented (total mice tested per group in two experiments n = 21). The arrow indicates the day when treatment was performed. Data are shown as mean ± SEM daily clinical score. (B) Quantification of the area under the curve (AUC) corresponding to the first relapse (days 15 to 27 after disease induction, as delineated by the two dotted lines on panel (A) during the course of the disease). Data are shown as mean ± SEM. ** p < 0.01.

Figure 2

Co-transplantation of HSCs and MSCs has no synergistic beneficial effect on EAE course or severity. (A) EAE course of mice untreated, irradiated and treated with HSCs, or irradiated and co-transplanted with HSCs and MSCs (total mice tested per group n = 13). The arrow indicates the day when treatment was performed. Data are shown as mean ± SEM of daily clinical score. (B) Quantification of the area under the curve (AUC) corresponding to the first relapse (days 13 to 23 after disease induction, as delineated by the two doted lines on panel (A) during the course of the disease). Data are shown as mean ± SEM. * p < 0.05. (C) Comparison of the mean of clinical score between groups of mice untreated, irradiated and treated with HSCs, or irradiated and co-transplanted with HSCs and MSCs, at disease peak (day 14). ns refers to “not-statistically significant”. Data are shown as mean ± SEM. ** p < 0.01, *** p < 0.001.

Figure 3

Injection of MSCs together with HSCs does not enhance immune cell engraftment. Quantification of immune cells performed by counting the absolute number/μL of EDTA-treated peripheral blood of T cells (CD3), monocyte/macrophages (CD11b) and B cells (CD19) at: (A) 3 days after TBI, (B) 12 days after TBI, and (C) 33 days after TBI. Quantitative data are presented as mean ± SEM of CD45-gated cells with n = 3 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Anti-inflammatory IL-4 is upregulated in co-transplanted mice. Quantification of (A) IL-4 and (B) IFN-γ in plasma from EAE-affected mice untreated, irradiated and treated with HSCs, or irradiated and co-transplanted with HSCs and MSCs, at 12 days post-treatment. Data are presented as mean ± SEM cytokine concentration of triplicate plasma samples of n = 4 mice per group. ** p < 0.01 *** p < 0.001.