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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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CXCL12-Ig suppresses ongoing EAE. (A) 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, 13, 15, and 17, these groups were injected i.v. with either PBS (open circles), CXCL12-Ig (closed circles), or β-actin–Ig (open squares) and were monitored for the 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. The arrow indicates the first day of CXCL12-Ig administration. (B) On day 20, three representative mice from each group were subjected to histological analysis of the lumbar spinal cord (eight sections per sample). A scale ranging from 0 to 3, based on the number of perivascular lesions per section, was used to quantify the histological score of disease, as described in Materials and methods. The table presents the quantification analysis of these sections, and a representative section from each group is also shown. Representative sections were also subjected to immunohistochemistry for IL-10. Arrows indicate cells stained positive for IL-10. Bars, 200 μm. (C) In a subsequent experiment, conducted under the same experimental protocol, mice were killed on day 15, and spleen cells from each group were cultured in the presence of their target antigen (MOGp35-55). After 24 h, levels of IL-10, IL-4, TGF-β, IL-12, IL-17, IL-23, and TNF-α were recorded by ELISA. Results are shown as the mean of triplicates ± SE. (D, a) C57BL/6 female mice were subjected to active induction of a long-term form of disease (reference 21), and just after the onset of disease they were separated into equally sick groups (n = 6 mice each). Twice a week, these groups were injected i.v. with PBS (closed circles), CXCL12-Ig (open circles), or β-actin–Ig (open squares) and 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. The arrow indicates the first day of CXCL12-Ig administration. (b) Just before the peak of disease (day 24), primary T cells from the cervical lymph nodes of PBS-, β-actin–Ig–, and CXCL12-Ig–treated mice were subjected to MOGp35-55-induced activation. The proliferative response and levels of IL-2 were recorded. (c) Apoptosis in CD4+ T cells in these cultures was determined by flow cytometry using Annexin V/PI staining (percentages are shown).
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fig4: CXCL12-Ig suppresses ongoing EAE. (A) 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, 13, 15, and 17, these groups were injected i.v. with either PBS (open circles), CXCL12-Ig (closed circles), or β-actin–Ig (open squares) and were monitored for the 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. The arrow indicates the first day of CXCL12-Ig administration. (B) On day 20, three representative mice from each group were subjected to histological analysis of the lumbar spinal cord (eight sections per sample). A scale ranging from 0 to 3, based on the number of perivascular lesions per section, was used to quantify the histological score of disease, as described in Materials and methods. The table presents the quantification analysis of these sections, and a representative section from each group is also shown. Representative sections were also subjected to immunohistochemistry for IL-10. Arrows indicate cells stained positive for IL-10. Bars, 200 μm. (C) In a subsequent experiment, conducted under the same experimental protocol, mice were killed on day 15, and spleen cells from each group were cultured in the presence of their target antigen (MOGp35-55). After 24 h, levels of IL-10, IL-4, TGF-β, IL-12, IL-17, IL-23, and TNF-α were recorded by ELISA. Results are shown as the mean of triplicates ± SE. (D, a) C57BL/6 female mice were subjected to active induction of a long-term form of disease (reference 21), and just after the onset of disease they were separated into equally sick groups (n = 6 mice each). Twice a week, these groups were injected i.v. with PBS (closed circles), CXCL12-Ig (open circles), or β-actin–Ig (open squares) and 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. The arrow indicates the first day of CXCL12-Ig administration. (b) Just before the peak of disease (day 24), primary T cells from the cervical lymph nodes of PBS-, β-actin–Ig–, and CXCL12-Ig–treated mice were subjected to MOGp35-55-induced activation. The proliferative response and levels of IL-2 were recorded. (c) Apoptosis in CD4+ T cells in these cultures was determined by flow cytometry using Annexin V/PI staining (percentages are shown).

Mentions: We then explored the ability of this fusion protein to suppress ongoing EAE. Fig. 4 A shows that repeated administration of this fusion protein, but not of a control fusion protein comprised from soluble β-actin–Ig, could effectively and rapidly suppress the disease. Although all control mice continued to develop a semichronic form of disease that persisted >4 wk, all CXCL12-Ig–treated mice went into remission within 7–8 d (day 20 control group mean maximal score of 2.1 ± 0.166 and 2.3 ± 0.26 vs. 0.166 ± 0.16; P < 0.001). Histological analysis conducted on lumbar spinal cord sections on day 20 verified the clinical results (mean histological score of 0.4 ± 0.3 vs. 2.3 ± 0.3 and 2.1 ± 0.3 in control groups). Representative sections were also subjected to immunohistochemical analysis of IL-10, showing the existence of IL-10–producing cells within the few perivascular infiltrates in sections from CXCL12-Ig–treated mice but not control groups (Fig. 4 B). In a subsequent set of experiments, spleen cells from these groups were cultured in the presence of their target antigen (MOGp35-55), and the levels of IL-10, IL-4, IL-12, TGF-β, IL-17, IL-23, and TNF-α were recorded (Fig. 4 C). The significantly higher levels of IL-10 detected in cultures from CXCL12-Ig–treated mice (1,450 ± 170 compared with 750 ± 65 and 790 ± 70 pg/ml; P < 0.01) was accompanied by a reduced production of macrophage proinflammatory mediators, including IL-12 (330 ± 27 vs. 920 ± 80 and 1,230 ± 140 pg/ml; P < 0.01), IL-23 (13 ± 1.2 vs. 30 ± 4.3 and 32 ± 3.1 pg/ml; P < 0.001), and TNF-α (780 ± 55 vs. 1,540 ± 130 and 1,420 ± 60 pg/ml; P < 0.01), which is also largely produced by Th1 cells (20). No significant changes in TGF-β or IL-4 levels were noted. Thus, therapy with CXCL12-Ig promotes antiinflammatory cytokine production, particularly IL-10, whereas blocking the production of proinflammatory cytokines, including those directing the polarization of Th1 and Th17 cells, particularly the cytokine IL-17 (160 ± 22 vs. 820 ± 115 and 780 ± 95 pg/ml; P < 0.001).


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)

CXCL12-Ig suppresses ongoing EAE. (A) 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, 13, 15, and 17, these groups were injected i.v. with either PBS (open circles), CXCL12-Ig (closed circles), or β-actin–Ig (open squares) and were monitored for the 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. The arrow indicates the first day of CXCL12-Ig administration. (B) On day 20, three representative mice from each group were subjected to histological analysis of the lumbar spinal cord (eight sections per sample). A scale ranging from 0 to 3, based on the number of perivascular lesions per section, was used to quantify the histological score of disease, as described in Materials and methods. The table presents the quantification analysis of these sections, and a representative section from each group is also shown. Representative sections were also subjected to immunohistochemistry for IL-10. Arrows indicate cells stained positive for IL-10. Bars, 200 μm. (C) In a subsequent experiment, conducted under the same experimental protocol, mice were killed on day 15, and spleen cells from each group were cultured in the presence of their target antigen (MOGp35-55). After 24 h, levels of IL-10, IL-4, TGF-β, IL-12, IL-17, IL-23, and TNF-α were recorded by ELISA. Results are shown as the mean of triplicates ± SE. (D, a) C57BL/6 female mice were subjected to active induction of a long-term form of disease (reference 21), and just after the onset of disease they were separated into equally sick groups (n = 6 mice each). Twice a week, these groups were injected i.v. with PBS (closed circles), CXCL12-Ig (open circles), or β-actin–Ig (open squares) and 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. The arrow indicates the first day of CXCL12-Ig administration. (b) Just before the peak of disease (day 24), primary T cells from the cervical lymph nodes of PBS-, β-actin–Ig–, and CXCL12-Ig–treated mice were subjected to MOGp35-55-induced activation. The proliferative response and levels of IL-2 were recorded. (c) Apoptosis in CD4+ T cells in these cultures was determined by flow cytometry using Annexin V/PI staining (percentages are shown).
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Related In: Results  -  Collection

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fig4: CXCL12-Ig suppresses ongoing EAE. (A) 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, 13, 15, and 17, these groups were injected i.v. with either PBS (open circles), CXCL12-Ig (closed circles), or β-actin–Ig (open squares) and were monitored for the 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. The arrow indicates the first day of CXCL12-Ig administration. (B) On day 20, three representative mice from each group were subjected to histological analysis of the lumbar spinal cord (eight sections per sample). A scale ranging from 0 to 3, based on the number of perivascular lesions per section, was used to quantify the histological score of disease, as described in Materials and methods. The table presents the quantification analysis of these sections, and a representative section from each group is also shown. Representative sections were also subjected to immunohistochemistry for IL-10. Arrows indicate cells stained positive for IL-10. Bars, 200 μm. (C) In a subsequent experiment, conducted under the same experimental protocol, mice were killed on day 15, and spleen cells from each group were cultured in the presence of their target antigen (MOGp35-55). After 24 h, levels of IL-10, IL-4, TGF-β, IL-12, IL-17, IL-23, and TNF-α were recorded by ELISA. Results are shown as the mean of triplicates ± SE. (D, a) C57BL/6 female mice were subjected to active induction of a long-term form of disease (reference 21), and just after the onset of disease they were separated into equally sick groups (n = 6 mice each). Twice a week, these groups were injected i.v. with PBS (closed circles), CXCL12-Ig (open circles), or β-actin–Ig (open squares) and 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. The arrow indicates the first day of CXCL12-Ig administration. (b) Just before the peak of disease (day 24), primary T cells from the cervical lymph nodes of PBS-, β-actin–Ig–, and CXCL12-Ig–treated mice were subjected to MOGp35-55-induced activation. The proliferative response and levels of IL-2 were recorded. (c) Apoptosis in CD4+ T cells in these cultures was determined by flow cytometry using Annexin V/PI staining (percentages are shown).
Mentions: We then explored the ability of this fusion protein to suppress ongoing EAE. Fig. 4 A shows that repeated administration of this fusion protein, but not of a control fusion protein comprised from soluble β-actin–Ig, could effectively and rapidly suppress the disease. Although all control mice continued to develop a semichronic form of disease that persisted >4 wk, all CXCL12-Ig–treated mice went into remission within 7–8 d (day 20 control group mean maximal score of 2.1 ± 0.166 and 2.3 ± 0.26 vs. 0.166 ± 0.16; P < 0.001). Histological analysis conducted on lumbar spinal cord sections on day 20 verified the clinical results (mean histological score of 0.4 ± 0.3 vs. 2.3 ± 0.3 and 2.1 ± 0.3 in control groups). Representative sections were also subjected to immunohistochemical analysis of IL-10, showing the existence of IL-10–producing cells within the few perivascular infiltrates in sections from CXCL12-Ig–treated mice but not control groups (Fig. 4 B). In a subsequent set of experiments, spleen cells from these groups were cultured in the presence of their target antigen (MOGp35-55), and the levels of IL-10, IL-4, IL-12, TGF-β, IL-17, IL-23, and TNF-α were recorded (Fig. 4 C). The significantly higher levels of IL-10 detected in cultures from CXCL12-Ig–treated mice (1,450 ± 170 compared with 750 ± 65 and 790 ± 70 pg/ml; P < 0.01) was accompanied by a reduced production of macrophage proinflammatory mediators, including IL-12 (330 ± 27 vs. 920 ± 80 and 1,230 ± 140 pg/ml; P < 0.01), IL-23 (13 ± 1.2 vs. 30 ± 4.3 and 32 ± 3.1 pg/ml; P < 0.001), and TNF-α (780 ± 55 vs. 1,540 ± 130 and 1,420 ± 60 pg/ml; P < 0.01), which is also largely produced by Th1 cells (20). No significant changes in TGF-β or IL-4 levels were noted. Thus, therapy with CXCL12-Ig promotes antiinflammatory cytokine production, particularly IL-10, whereas blocking the production of proinflammatory cytokines, including those directing the polarization of Th1 and Th17 cells, particularly the cytokine IL-17 (160 ± 22 vs. 820 ± 115 and 780 ± 95 pg/ml; P < 0.001).

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