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Results: 4

1.
Figure 1

Figure 1. Laquinimod prevents EAE and decreases encephalitogenicity of T cells.. From: Laquinimod, a Quinoline-3-Carboxamide, Induces Type II Myeloid Cells That Modulate Central Nervous System Autoimmunity.

DBA/1 (A) mice and C57BL/6 mice (B) were treated daily with laquinimod (25 mg/kg, n = 6/group) or vehicle (water, n = 6/group) starting from the day of immunization with rMOG 1-125 (DBA/1) or MOG p35-55 (C57BL/6). Fourteen days after immunization, CNS infiltrating cells (C) and spleen cells (D) from C57BL/6 mice treated with laquinimod or vehicle were evaluated for secretion of IFN-γ, IL-17, GM-CSF and for expression of CD25 and Foxp3 by CD4+ cells. Representative FACS staining is shown including quantification (n = 4–5 mice per group). (E) Laquinimod decreases pathogenic potential of T cells. Splenocytes and lymph node cells were isolated from PLP-immunized mice treated with laquinimod or vehicle. Cells were restimulated in vitro, and 107 cells were transferred into naïve SJL/J recipients (n = 5/group). Data are representative of two independent experiments. For EAE disease course, mean disease score ± s.e.m. are displayed; for other experiments mean ± s.d. *P<0.05, **P<0.01, Mann-Whitney U test.

Ulf Schulze-Topphoff, et al. PLoS One. 2012;7(3):e33797.
2.
Figure 3

Figure 3. Laquinimod alters myeloid APC subsets and inhibits Th1 and Th17 polarization of myelin-specific T cells.. From: Laquinimod, a Quinoline-3-Carboxamide, Induces Type II Myeloid Cells That Modulate Central Nervous System Autoimmunity.

All experiments were conducted with myeloid-APC purified (purity ≥95%) by MACS sorting from mice treated with laquinimod or vehicle for ten days. (A) DC were isolated from the spleen and defined as CD11chigh. Percentages of splenic CD11chigh CD4+ cDC (CD4+CD11b+) from laquinimod-treated or vehicle-treated mice (n = 4), and percentage of total splenic CD4+ cells from laquinimod- and vehicle-treated mice (n = 5) are shown. (B) Splenic and blood CD11b+ cells were stained for Gr1. Relative percentages of CD11b+Gr1hi (arrow points to Gr1hi cells) out of total splenic and blood CD11b+ cells from mice treated with laquinimod (dark grey bar) or vehicle (white bar) is shown (n = 4). (C–E) Purified splenic CD11b+, CD11b+CD11c (C, D) or CD11c+ (E) cells were used as APC in co-culture with naïve CD4+ 2D2 T cells and Ag (MOG p35-55). Th1 and Th17 differentiation were induced as described above. Intracellular cytokine staining shows the percentage of IL-17 and IFN-γ after 3 days in culture (gate). (F) Purified splenic CD11b+CD11c or CD11c+ cells were used as APC in co-culture with naïve CD4+ 2D2 T cells under non-polarizing conditions. FACS analyses after four days in culture for IL-17 and IFN-γ and for Foxp3 by CD4+ cells is shown. Data shown as mean ± s.e.m. are representative of at least two independent experiments; *P<0.05, Mann-Whitney U test.

Ulf Schulze-Topphoff, et al. PLoS One. 2012;7(3):e33797.
3.
Figure 2

Figure 2. Laquinimod reverses RR-EAE and inhibits inflammatory T cell responses via a direct effect on myeloid APC.. From: Laquinimod, a Quinoline-3-Carboxamide, Induces Type II Myeloid Cells That Modulate Central Nervous System Autoimmunity.

(A) Daily oral laquinimod treatment reverses relapsing remitting EAE. SJL/J mice were immunized with PLP p139-151 and treated with laquinimod or vehicle (n = 9) at the remission phase (arrow points to the start of the treatment). (B) Lesion quantification showed reduced total number of meningeal and parenchymal inflammatory foci in SJL/J mice treated with laquinimod after first exacerbation of the disease. Representative Luxol fast blue-H&E staining of the cerebellum is shown. (C, D) Laquinimod-treated myeloid APC inhibit differentiation of naive T cells into Th1 and Th17 cells. Whole splenic in vivo laquinimod-treated or untreated CD11b+ cells were used as APC in co-culture with untreated naive (CD4+CD44CD62L+) T cells from MOG p35-55 TCR-transgenic mice (2D2). Conversely, naive T cells were isolated from laquinimod- or vehicle-treated 2D2 mice and cultured with purified vehicle-treated CD11b+ cells and Ag (MOG p35-55). Polarization of naïve T cells into Th1 lineage was induced by IL-12 (C) and polarization into Th17 lineage was induced by IL-23, IL-6 and TGF-β (D). Intracellular cytokine staining for IFN-γ and IL-17 after three days in culture is shown. For all experiments, data shown are representative of two independent experiments. For EAE disease course and other experiments, mean disease score and mean ± s.e.m. are displayed; *P<0.05, Mann-Whitney U test.

Ulf Schulze-Topphoff, et al. PLoS One. 2012;7(3):e33797.
4.
Figure 4

Figure 4. Laquinimod-induced type II (M2) monocytes reverse established EAE.. From: Laquinimod, a Quinoline-3-Carboxamide, Induces Type II Myeloid Cells That Modulate Central Nervous System Autoimmunity.

Laquinimod-induced anti-inflammatory cytokine shift in CD11b+CD11c and CD11c+ cells. (A) FACS analysis of intracellular production of TNF, IL12/23p40, IL-6 and IL-10 by CD11b+CD11c and CD11c+ cells isolated from spleens of naive (unimmunized) mice treated with laquinimod or vehicle. (B) Cell surface FACS analysis of MHC II, proinflammatory and inhibitory costimulatory molecules on CD11b+CD11c cells. (C) In vivo laquinimod treatment affects signaling pathways that participate in proinflammatory cytokine production. Protein extracts were isolated from peritoneal macrophages of naïve C57BL/6 mice treated with laquinimod or vehicle and stimulated with LPS for various time points. Phosphorylated (P) STAT1, (P) p38-MAPK, Pan-STAT1 and Pan-p38-MAPK were detected by Western blot analysis. (D) CD11b+ cells from laquinimod-treated donor mice reversed established EAE. 5×106 purified splenic CD11b+ cells from mice treated with laquinimod or vehicle were injected i.v. into recipient C57BL/6 mice immunized with MOG p35–55 after they developed a disease grade of 2 (black arrow indicates time point of adoptive transfer, (n = 5/group). (E) Quantification showed reduced total number of inflammatory foci after adoptive transfer of in vivo laquinimod treated CD11b+ cells into C57BL/6 mice immunized with MOG p35-55. Data shown in panels above are representative of three independent experiments. For EAE disease course, mean disease score ± s.e.m. are displayed; *P<0.05, **P<0.01 Mann-Whitney U test.

Ulf Schulze-Topphoff, et al. PLoS One. 2012;7(3):e33797.

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