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CXCL12 (SDF-1alpha) suppresses ongoing experimental autoimmune encephalomyelitis by selecting antigen-specific regulatory T cells.

Meiron M, Zohar Y, Anunu R, Wildbaum G, Karin N - J. Exp. Med. (2008)

Bottom Line: The beneficial effect included selection of antigen-specific T cells that were CD4(+)CD25(-)Foxp3(-)IL-10(high), which could adoptively transfer disease resistance, and suppression of Th17 selection.However, in vitro functional analysis of these cells suggested that, even though CXCL12-Ig-induced tolerance is IL-10 dependent, IL-10-independent mechanisms may also contribute to their regulatory function.Collectively, our results not only demonstrate, for the first time, that a chemokine functions as a regulatory mediator, but also suggest a novel way for treating multiple sclerosis and possibly other inflammatory autoimmune diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel.

ABSTRACT
Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the central nervous system induced by antigen-specific effector Th17 and Th1 cells. We show that a key chemokine, CXCL12 (stromal cell-derived factor 1alpha), redirects the polarization of effector Th1 cells into CD4(+)CD25(-)Foxp3(-)interleukin (IL) 10(high) antigen-specific regulatory T cells in a CXCR4-dependent manner, and by doing so acts as a regulatory mediator restraining the autoimmune inflammatory process. In an attempt to explore the therapeutic implication of these findings, we have generated a CXCL12-immunoglobulin (Ig) fusion protein that, when administered during ongoing EAE, rapidly suppresses the disease in wild-type but not IL-10-deficient mice. Anti-IL-10 neutralizing antibodies could reverse this suppression. The beneficial effect included selection of antigen-specific T cells that were CD4(+)CD25(-)Foxp3(-)IL-10(high), which could adoptively transfer disease resistance, and suppression of Th17 selection. However, in vitro functional analysis of these cells suggested that, even though CXCL12-Ig-induced tolerance is IL-10 dependent, IL-10-independent mechanisms may also contribute to their regulatory function. Collectively, our results not only demonstrate, for the first time, that a chemokine functions as a regulatory mediator, but also suggest a novel way for treating multiple sclerosis and possibly other inflammatory autoimmune diseases.

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Related in: MedlinePlus

Antigen-specific T cells selected in the presence of CXCL12 suppress EAE. C57BL/6 female mice were subjected to active induction of EAE (MOGp35-55/CFA), and just after the onset of disease (day 11), they were separated into equally sick groups (n = 6 mice each). On days 11 and 13, these groups were injected i.v. either with PBS, CXCL12-Ig, or β-actin–Ig. On day 15, the spleens were removed. (A) Spleen sections were subjected to immunohistochemical analysis for IL-10 expression. Bars, 200 μm. (B) Spleen cells from the different groups were cultured with the target antigen for 72 h and were then subjected to flow cytometry analysis for intracellular staining of IL-10 in macrophages/dendritic cells (CD14+) and in CD4+ T cells (percentages are shown). (C) Spleen cells isolated from treated mice (in B) were subjected to antigen-specific activation and were injected (20 × 106 cells per mouse) into recipient EAE mice at the onset of disease (n = 6 mice per group) either with cells isolated from CXCL12-Ig mice (closed squares) or from β-actin–Ig–treated EAE mice (closed circles). A third group of recipients was administered with PBS (open squares). All groups were monitored for the development and progression of disease by an observer blind to the experimental protocol. Results of one out of three independent experiments (n = 6 mice per each group) are shown as the mean maximal score ± SE. (D) Before being administered to EAE mice (in C), IL-10high T cells selected in CXCL12-Ig–treated mice were tested for the expression of CD25 and FOXp3 (percentages are shown). (E) Spleen cells from EAE mice that were treated with CXCL12-Ig, as described in C, were subjected to antigen-specific in vitro activation and separated into CD4+ and CD14+ (MACS beads). 10 × 106 cells per mouse were injected into recipient EAE mice at the onset of disease (n = 6 mice per group) as follows: CD4+ cells isolated from CXCL12-Ig mice (open circles) and CD14+ cells isolated from CXCL12-Ig mice (closed circles). Control EAE mice were administered with PBS (closed squares). All groups were monitored for the development and progression of disease by an observer blind to the experimental protocol. Results of one out of three independent experiments (n = 6 mice per each group) are shown as the mean maximal score ± SE. (F) IL-10high T cells selected in CXCL12-Ig treated mice were tested for their ability to suppress the proliferative response of antigen-specific effector T cells from control EAE mice when added at different effector/regulatory ratios (shaded bars) and in the presence of 50 μg/ml of neutralizing anti–IL-10 antibody (open bars). (G) IL-10−/− mice (a) and C57BL/6 mice (b) were subjected to the treatment protocol described in Fig. 4 A. On day 13, mice were injected with either PBS (open squares), β-actin–Ig (closed circles), or CXCL12-Ig (closed squares). (c) C57BL/6 female mice were subjected to active induction of EAE, and just after the onset of disease (day 9), they were separated into equally sick groups (n = 6 mice each). On days 10, 12, and 14, mice were injected i.v. with PBS (open squares), CXCL12-Ig (closed circles), anti–IL-10 mAb (closed squares), or CXCL12-Ig followed by anti–IL-10 mAb injected 6 h later (open triangles). Mice were monitored daily for the progression of the disease by an observer blind to the treatment protocol. The arrow indicates the first day of CXCL12-Ig administration. Results of one out of three independent experiments with similar results (n = 6 mice per each group) are shown as the mean maximal score ± SE.
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fig5: Antigen-specific T cells selected in the presence of CXCL12 suppress EAE. C57BL/6 female mice were subjected to active induction of EAE (MOGp35-55/CFA), and just after the onset of disease (day 11), they were separated into equally sick groups (n = 6 mice each). On days 11 and 13, these groups were injected i.v. either with PBS, CXCL12-Ig, or β-actin–Ig. On day 15, the spleens were removed. (A) Spleen sections were subjected to immunohistochemical analysis for IL-10 expression. Bars, 200 μm. (B) Spleen cells from the different groups were cultured with the target antigen for 72 h and were then subjected to flow cytometry analysis for intracellular staining of IL-10 in macrophages/dendritic cells (CD14+) and in CD4+ T cells (percentages are shown). (C) Spleen cells isolated from treated mice (in B) were subjected to antigen-specific activation and were injected (20 × 106 cells per mouse) into recipient EAE mice at the onset of disease (n = 6 mice per group) either with cells isolated from CXCL12-Ig mice (closed squares) or from β-actin–Ig–treated EAE mice (closed circles). A third group of recipients was administered with PBS (open squares). All groups were monitored for the development and progression of disease by an observer blind to the experimental protocol. Results of one out of three independent experiments (n = 6 mice per each group) are shown as the mean maximal score ± SE. (D) Before being administered to EAE mice (in C), IL-10high T cells selected in CXCL12-Ig–treated mice were tested for the expression of CD25 and FOXp3 (percentages are shown). (E) Spleen cells from EAE mice that were treated with CXCL12-Ig, as described in C, were subjected to antigen-specific in vitro activation and separated into CD4+ and CD14+ (MACS beads). 10 × 106 cells per mouse were injected into recipient EAE mice at the onset of disease (n = 6 mice per group) as follows: CD4+ cells isolated from CXCL12-Ig mice (open circles) and CD14+ cells isolated from CXCL12-Ig mice (closed circles). Control EAE mice were administered with PBS (closed squares). All groups were monitored for the development and progression of disease by an observer blind to the experimental protocol. Results of one out of three independent experiments (n = 6 mice per each group) are shown as the mean maximal score ± SE. (F) IL-10high T cells selected in CXCL12-Ig treated mice were tested for their ability to suppress the proliferative response of antigen-specific effector T cells from control EAE mice when added at different effector/regulatory ratios (shaded bars) and in the presence of 50 μg/ml of neutralizing anti–IL-10 antibody (open bars). (G) IL-10−/− mice (a) and C57BL/6 mice (b) were subjected to the treatment protocol described in Fig. 4 A. On day 13, mice were injected with either PBS (open squares), β-actin–Ig (closed circles), or CXCL12-Ig (closed squares). (c) C57BL/6 female mice were subjected to active induction of EAE, and just after the onset of disease (day 9), they were separated into equally sick groups (n = 6 mice each). On days 10, 12, and 14, mice were injected i.v. with PBS (open squares), CXCL12-Ig (closed circles), anti–IL-10 mAb (closed squares), or CXCL12-Ig followed by anti–IL-10 mAb injected 6 h later (open triangles). Mice were monitored daily for the progression of the disease by an observer blind to the treatment protocol. The arrow indicates the first day of CXCL12-Ig administration. Results of one out of three independent experiments with similar results (n = 6 mice per each group) are shown as the mean maximal score ± SE.

Mentions: The suppression of EAE after CXCL12-Ig therapy could result from the reduced production of proinflammatory mediators by macrophages, including those selecting Th17 and Th1 effector T cells (IL-23 and IL-12; Fig. 4 C), and/or from possible selection of antigen-specific regulatory T cells, potentially capable of suppressing an ongoing disease in adoptive transfer experiments. To explore this possibility, mice were subjected to active induction of EAE and then to the administration of CXCL12-Ig, β-actin–Ig, or PBS, as described in the legend to Fig. 4 A. On day 15, when the therapeutic effect of CXCL12-Ig was highly significant (Fig. 4 A), spleens were removed. Immunohistochemical analysis of representative sections revealed high IL-10 expression in spleen sections from CXCL12-Ig–treated mice (Fig. 5 A). Intracellular flow cytometry analysis, conducted on samples of cultured cells from these groups, clearly showed a significant increase in IL-10high CD4+ T cells (4.2 vs. 1.1 and 0.9%, respectively; Fig. 5 B), as well as in IL-10high CD14+ macrophages/dendritic cells (7.7 vs. 4.8 and 4.9%, respectively) in the CXCL12-Ig–treated mice. T cells from donors treated with protective CXCL12-Ig or β-actin–Ig were then administered to mice suffering from active EAE. After antigen-specific activation, these cells were administered to EAE recipients (just after the onset of disease). Fig. 5 C shows that although the administration of spleen cells from EAE donors, treated with β-actin–Ig, aggravated the severity of disease (day 18 mean score of 5 ± 0 vs. 3 ± 0.26; P < 0.01), the administration of spleen cells from CXCL12-Ig–treated mice led to a rapid recovery (day 18 mean score of 0 ± 0; P < 0.001). Further analysis of the transferred cells showed that the vast majority of IL-10–producing T cells from protected donors were Foxp3− (96%), CD25− (86%; Fig. 5 D). In an attempt to elucidate the possibility that these cells direct disease suppression, we have repeated the adoptive transfer experiment described in Fig. 5 C. Hence, spleen cells from EAE mice treated with CXCL12-Ig were separated (MACS beads, negative selection) to either CD4+ or CD14+ cells, and only then were they injected into recipient EAE mice (10 × 106 cells per mouse). Our results clearly show that under these conditions only CD4+ T cells could effectively (P < 0.01) suppress the disease (Fig. 5 E). Thus, CXCL12-Ig selects antigen–specific regulatory CD4+ T cells that are IL-10highCD25−Foxp3−, which are capable of suppressing EAE in adoptive transfer experiments.


CXCL12 (SDF-1alpha) suppresses ongoing experimental autoimmune encephalomyelitis by selecting antigen-specific regulatory T cells.

Meiron M, Zohar Y, Anunu R, Wildbaum G, Karin N - J. Exp. Med. (2008)

Antigen-specific T cells selected in the presence of CXCL12 suppress EAE. C57BL/6 female mice were subjected to active induction of EAE (MOGp35-55/CFA), and just after the onset of disease (day 11), they were separated into equally sick groups (n = 6 mice each). On days 11 and 13, these groups were injected i.v. either with PBS, CXCL12-Ig, or β-actin–Ig. On day 15, the spleens were removed. (A) Spleen sections were subjected to immunohistochemical analysis for IL-10 expression. Bars, 200 μm. (B) Spleen cells from the different groups were cultured with the target antigen for 72 h and were then subjected to flow cytometry analysis for intracellular staining of IL-10 in macrophages/dendritic cells (CD14+) and in CD4+ T cells (percentages are shown). (C) Spleen cells isolated from treated mice (in B) were subjected to antigen-specific activation and were injected (20 × 106 cells per mouse) into recipient EAE mice at the onset of disease (n = 6 mice per group) either with cells isolated from CXCL12-Ig mice (closed squares) or from β-actin–Ig–treated EAE mice (closed circles). A third group of recipients was administered with PBS (open squares). All groups were monitored for the development and progression of disease by an observer blind to the experimental protocol. Results of one out of three independent experiments (n = 6 mice per each group) are shown as the mean maximal score ± SE. (D) Before being administered to EAE mice (in C), IL-10high T cells selected in CXCL12-Ig–treated mice were tested for the expression of CD25 and FOXp3 (percentages are shown). (E) Spleen cells from EAE mice that were treated with CXCL12-Ig, as described in C, were subjected to antigen-specific in vitro activation and separated into CD4+ and CD14+ (MACS beads). 10 × 106 cells per mouse were injected into recipient EAE mice at the onset of disease (n = 6 mice per group) as follows: CD4+ cells isolated from CXCL12-Ig mice (open circles) and CD14+ cells isolated from CXCL12-Ig mice (closed circles). Control EAE mice were administered with PBS (closed squares). All groups were monitored for the development and progression of disease by an observer blind to the experimental protocol. Results of one out of three independent experiments (n = 6 mice per each group) are shown as the mean maximal score ± SE. (F) IL-10high T cells selected in CXCL12-Ig treated mice were tested for their ability to suppress the proliferative response of antigen-specific effector T cells from control EAE mice when added at different effector/regulatory ratios (shaded bars) and in the presence of 50 μg/ml of neutralizing anti–IL-10 antibody (open bars). (G) IL-10−/− mice (a) and C57BL/6 mice (b) were subjected to the treatment protocol described in Fig. 4 A. On day 13, mice were injected with either PBS (open squares), β-actin–Ig (closed circles), or CXCL12-Ig (closed squares). (c) C57BL/6 female mice were subjected to active induction of EAE, and just after the onset of disease (day 9), they were separated into equally sick groups (n = 6 mice each). On days 10, 12, and 14, mice were injected i.v. with PBS (open squares), CXCL12-Ig (closed circles), anti–IL-10 mAb (closed squares), or CXCL12-Ig followed by anti–IL-10 mAb injected 6 h later (open triangles). Mice were monitored daily for the progression of the disease by an observer blind to the treatment protocol. The arrow indicates the first day of CXCL12-Ig administration. Results of one out of three independent experiments with similar results (n = 6 mice per each group) are shown as the mean maximal score ± SE.
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fig5: Antigen-specific T cells selected in the presence of CXCL12 suppress EAE. C57BL/6 female mice were subjected to active induction of EAE (MOGp35-55/CFA), and just after the onset of disease (day 11), they were separated into equally sick groups (n = 6 mice each). On days 11 and 13, these groups were injected i.v. either with PBS, CXCL12-Ig, or β-actin–Ig. On day 15, the spleens were removed. (A) Spleen sections were subjected to immunohistochemical analysis for IL-10 expression. Bars, 200 μm. (B) Spleen cells from the different groups were cultured with the target antigen for 72 h and were then subjected to flow cytometry analysis for intracellular staining of IL-10 in macrophages/dendritic cells (CD14+) and in CD4+ T cells (percentages are shown). (C) Spleen cells isolated from treated mice (in B) were subjected to antigen-specific activation and were injected (20 × 106 cells per mouse) into recipient EAE mice at the onset of disease (n = 6 mice per group) either with cells isolated from CXCL12-Ig mice (closed squares) or from β-actin–Ig–treated EAE mice (closed circles). A third group of recipients was administered with PBS (open squares). All groups were monitored for the development and progression of disease by an observer blind to the experimental protocol. Results of one out of three independent experiments (n = 6 mice per each group) are shown as the mean maximal score ± SE. (D) Before being administered to EAE mice (in C), IL-10high T cells selected in CXCL12-Ig–treated mice were tested for the expression of CD25 and FOXp3 (percentages are shown). (E) Spleen cells from EAE mice that were treated with CXCL12-Ig, as described in C, were subjected to antigen-specific in vitro activation and separated into CD4+ and CD14+ (MACS beads). 10 × 106 cells per mouse were injected into recipient EAE mice at the onset of disease (n = 6 mice per group) as follows: CD4+ cells isolated from CXCL12-Ig mice (open circles) and CD14+ cells isolated from CXCL12-Ig mice (closed circles). Control EAE mice were administered with PBS (closed squares). All groups were monitored for the development and progression of disease by an observer blind to the experimental protocol. Results of one out of three independent experiments (n = 6 mice per each group) are shown as the mean maximal score ± SE. (F) IL-10high T cells selected in CXCL12-Ig treated mice were tested for their ability to suppress the proliferative response of antigen-specific effector T cells from control EAE mice when added at different effector/regulatory ratios (shaded bars) and in the presence of 50 μg/ml of neutralizing anti–IL-10 antibody (open bars). (G) IL-10−/− mice (a) and C57BL/6 mice (b) were subjected to the treatment protocol described in Fig. 4 A. On day 13, mice were injected with either PBS (open squares), β-actin–Ig (closed circles), or CXCL12-Ig (closed squares). (c) C57BL/6 female mice were subjected to active induction of EAE, and just after the onset of disease (day 9), they were separated into equally sick groups (n = 6 mice each). On days 10, 12, and 14, mice were injected i.v. with PBS (open squares), CXCL12-Ig (closed circles), anti–IL-10 mAb (closed squares), or CXCL12-Ig followed by anti–IL-10 mAb injected 6 h later (open triangles). Mice were monitored daily for the progression of the disease by an observer blind to the treatment protocol. The arrow indicates the first day of CXCL12-Ig administration. Results of one out of three independent experiments with similar results (n = 6 mice per each group) are shown as the mean maximal score ± SE.
Mentions: The suppression of EAE after CXCL12-Ig therapy could result from the reduced production of proinflammatory mediators by macrophages, including those selecting Th17 and Th1 effector T cells (IL-23 and IL-12; Fig. 4 C), and/or from possible selection of antigen-specific regulatory T cells, potentially capable of suppressing an ongoing disease in adoptive transfer experiments. To explore this possibility, mice were subjected to active induction of EAE and then to the administration of CXCL12-Ig, β-actin–Ig, or PBS, as described in the legend to Fig. 4 A. On day 15, when the therapeutic effect of CXCL12-Ig was highly significant (Fig. 4 A), spleens were removed. Immunohistochemical analysis of representative sections revealed high IL-10 expression in spleen sections from CXCL12-Ig–treated mice (Fig. 5 A). Intracellular flow cytometry analysis, conducted on samples of cultured cells from these groups, clearly showed a significant increase in IL-10high CD4+ T cells (4.2 vs. 1.1 and 0.9%, respectively; Fig. 5 B), as well as in IL-10high CD14+ macrophages/dendritic cells (7.7 vs. 4.8 and 4.9%, respectively) in the CXCL12-Ig–treated mice. T cells from donors treated with protective CXCL12-Ig or β-actin–Ig were then administered to mice suffering from active EAE. After antigen-specific activation, these cells were administered to EAE recipients (just after the onset of disease). Fig. 5 C shows that although the administration of spleen cells from EAE donors, treated with β-actin–Ig, aggravated the severity of disease (day 18 mean score of 5 ± 0 vs. 3 ± 0.26; P < 0.01), the administration of spleen cells from CXCL12-Ig–treated mice led to a rapid recovery (day 18 mean score of 0 ± 0; P < 0.001). Further analysis of the transferred cells showed that the vast majority of IL-10–producing T cells from protected donors were Foxp3− (96%), CD25− (86%; Fig. 5 D). In an attempt to elucidate the possibility that these cells direct disease suppression, we have repeated the adoptive transfer experiment described in Fig. 5 C. Hence, spleen cells from EAE mice treated with CXCL12-Ig were separated (MACS beads, negative selection) to either CD4+ or CD14+ cells, and only then were they injected into recipient EAE mice (10 × 106 cells per mouse). Our results clearly show that under these conditions only CD4+ T cells could effectively (P < 0.01) suppress the disease (Fig. 5 E). Thus, CXCL12-Ig selects antigen–specific regulatory CD4+ T cells that are IL-10highCD25−Foxp3−, which are capable of suppressing EAE in adoptive transfer experiments.

Bottom Line: The beneficial effect included selection of antigen-specific T cells that were CD4(+)CD25(-)Foxp3(-)IL-10(high), which could adoptively transfer disease resistance, and suppression of Th17 selection.However, in vitro functional analysis of these cells suggested that, even though CXCL12-Ig-induced tolerance is IL-10 dependent, IL-10-independent mechanisms may also contribute to their regulatory function.Collectively, our results not only demonstrate, for the first time, that a chemokine functions as a regulatory mediator, but also suggest a novel way for treating multiple sclerosis and possibly other inflammatory autoimmune diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Bruce Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel.

ABSTRACT
Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the central nervous system induced by antigen-specific effector Th17 and Th1 cells. We show that a key chemokine, CXCL12 (stromal cell-derived factor 1alpha), redirects the polarization of effector Th1 cells into CD4(+)CD25(-)Foxp3(-)interleukin (IL) 10(high) antigen-specific regulatory T cells in a CXCR4-dependent manner, and by doing so acts as a regulatory mediator restraining the autoimmune inflammatory process. In an attempt to explore the therapeutic implication of these findings, we have generated a CXCL12-immunoglobulin (Ig) fusion protein that, when administered during ongoing EAE, rapidly suppresses the disease in wild-type but not IL-10-deficient mice. Anti-IL-10 neutralizing antibodies could reverse this suppression. The beneficial effect included selection of antigen-specific T cells that were CD4(+)CD25(-)Foxp3(-)IL-10(high), which could adoptively transfer disease resistance, and suppression of Th17 selection. However, in vitro functional analysis of these cells suggested that, even though CXCL12-Ig-induced tolerance is IL-10 dependent, IL-10-independent mechanisms may also contribute to their regulatory function. Collectively, our results not only demonstrate, for the first time, that a chemokine functions as a regulatory mediator, but also suggest a novel way for treating multiple sclerosis and possibly other inflammatory autoimmune diseases.

Show MeSH
Related in: MedlinePlus