Journal: Nature immunology

Article Title: Defective sphingosine-1-phosphate receptor 1 (S1P 1 ) phosphorylation exacerbates T H 17-mediated autoimmune neuroinflammation

doi: 10.1038/ni.2730

Figure Lengend Snippet: ( a ) Mean clinical score (± S. E. M.) of MOG 35–55 –immunized S1P 1 (S5A)(mice carrying phosphorylation defective S1pr1 gene) and WT (C57BL/6J) EAE mice (females, 8–9 weeks old). Immune cell proliferation (measured by 3 H[thymidine] incorporation) ( b ) and cytokine expression (measured by ELISA) ( c ) of ex vivo recall assay from MOG 35–55 -immunized S1P 1 (S5A)and WT EAE splenocytes (day 8 post-immunization). MOG; myelin oligodendrocyte glycoprotein, c.p.m.; counts per minute. ( d ) Photomicrograph (Luxol Fast Blue, Hematoxylin stain) and ( e ) quantification of CNS inflammation in WT (top) and S1P 1 (S5A) (bottom) EAE mice from ( a ). * p <0.05, ** p <0.01, Mann-Whitney U -test ( a ) and Student’s t -test ( b, c and e ). n =9–10/arm ( a ) and 3–5/arm ( b–e ). These experiments were repeated 3 times.

Article Snippet: WT C57BL/6J or S1P 1 (S5A) mice (female, 8–9 weeks old) were immunized with CFA and MOG 35–55, splenocytes and lymph node cells were harvested on day 9 post-immunization and expanded in vitro with MOG 10 μg/ml and IL-12 (20 ng/ml)(R&D Systems) for 72 h . Cells were then harvested, washed once with pre-warmed PBS, counted and injected into 5–6 weeks old Rag1 −/− naïve recipient mice (1×10 7 cells per mouse) intraperitoneally and followed clinically up to at least day 30.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Ex Vivo, Staining, MANN-WHITNEY

Journal: Nature immunology

Article Title: Defective sphingosine-1-phosphate receptor 1 (S1P 1 ) phosphorylation exacerbates T H 17-mediated autoimmune neuroinflammation

doi: 10.1038/ni.2730

Figure Lengend Snippet: ( a ) Mean clinical scores ((±S. E. M.) of Rag1 −/− adoptive transfer recipients of encephalitogenic cells from MOG 35–55 -immunized S1P 1 (S5A)and WT (C57BL/6J) EAE mice. Donors, n =10, females, 8–9 weeks old; recipients, n =5, females, 5–6 weeks old. ( b ) Quantification of total CNS infiltrating immune cells, ( c ) CD4 + and ( d, e ) CD4 + IL-17 + cells from CNS of S1P 1 (S5A)and WT EAE mice (at peak disease, days 13–15). Immunoblot depicting p-STAT3 ( f ), p-p38, p38 and IκBα ( h ) expression in splenocytes of S1Ps 1 (S5A) and WT EAE mice (day8, post-immunization). ( g ) Quantification of relative p-STAT3 expression [form ( f )]. ( b-g ) n =3–5 mice/arm. * p <0.05, Mann-Whitney U -test ( a ) and Student’s t- test ( b–d ). These experiments were repeated twice ( a ) and at least 3 times ( b–g ).

Article Snippet: WT C57BL/6J or S1P 1 (S5A) mice (female, 8–9 weeks old) were immunized with CFA and MOG 35–55, splenocytes and lymph node cells were harvested on day 9 post-immunization and expanded in vitro with MOG 10 μg/ml and IL-12 (20 ng/ml)(R&D Systems) for 72 h . Cells were then harvested, washed once with pre-warmed PBS, counted and injected into 5–6 weeks old Rag1 −/− naïve recipient mice (1×10 7 cells per mouse) intraperitoneally and followed clinically up to at least day 30.

Techniques: Adoptive Transfer Assay, Western Blot, Expressing, MANN-WHITNEY

Journal: Nature immunology

Article Title: Defective sphingosine-1-phosphate receptor 1 (S1P 1 ) phosphorylation exacerbates T H 17-mediated autoimmune neuroinflammation

doi: 10.1038/ni.2730

Figure Lengend Snippet: ( a ) Immunoblot depicting p-STAT3 expression in WT and S5A [S1P 1 (S5A)] EAE splenocytes (day 8 post-immunization) following in vitro activation with S1P (100nM) or W146 (S1P 1 antagonist) (20nM). ( b ) Quantification of normalized p-STAT3 expression from ( a ). ( c ) Cytokine expression in splenocyte cultures of MOG 35–55 -immunized WT EAE mice treated in vivo with S1P lyase inhibitor, THI (6.25mg/kg, daily intraperitoneal injections). IL-17 expression in CD3 + cells (activated with anti-CD3/anti-CD28) from ( d ) S1P 1 (S5A) naive mice treated in vitro with W146 (0–10nM), ( e ) WT naïve mice, treated in vitro with scrambled control (Ctrl) or S1pr1 -specific ( S1pr1 ) siRNA, or ( h ) naïve S1pr1 +/+ (WT) or S1pr1 −/− ( S1pr1 f/f Rosa26-CreER T2 ) mice. ( f and g ) Flow cytometric analysis of S1P 1 expression following treatment with S1pr1 or Ctrl siRNA treatment. * p <0.05, ** p <0.01, Student’s t -test. c–e and h ; analyzed by ELISA. ( a, b, d–g ) were performed 3–5 times, ( c and h ) twice. These experiments were performed with n =3–5 mice/arm. S1P; sphingosine-1-phosphate, W146; S1P 1 -specific antagonist, THI; 2-Acetyl-5-tetrahydroxybutyl Imidazole.

Article Snippet: WT C57BL/6J or S1P 1 (S5A) mice (female, 8–9 weeks old) were immunized with CFA and MOG 35–55, splenocytes and lymph node cells were harvested on day 9 post-immunization and expanded in vitro with MOG 10 μg/ml and IL-12 (20 ng/ml)(R&D Systems) for 72 h . Cells were then harvested, washed once with pre-warmed PBS, counted and injected into 5–6 weeks old Rag1 −/− naïve recipient mice (1×10 7 cells per mouse) intraperitoneally and followed clinically up to at least day 30.

Techniques: Western Blot, Expressing, In Vitro, Activation Assay, In Vivo, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Defective sphingosine-1-phosphate receptor 1 (S1P 1 ) phosphorylation exacerbates T H 17-mediated autoimmune neuroinflammation

doi: 10.1038/ni.2730

Figure Lengend Snippet: ( a ) IL-17 expression in culture supernatants of mixed lymphocyte cultures [CD3 + cells from MOG 35–55 -immunized S1P 1 (S5A) (S5A T) or C57BL/6J WT (WT T) EAE mice co-cultured with naïve, irradiated antigen presenting cells (APCs)from S1P 1 (S5A) (S5A APC) or WT (WT APC) in the presence of MOG 35–55 peptide] measured by ELISA. ( b ) IL-6 expression in an ex vivo recall assay of splenocyte cultures from MOG 35–55 -imunized S5A [S1P 1 (S5A)]and WT EAE mice (day 8 post-immunization). ( c ) Immunoblot depicting p-STAT3 expression in splenocytes from WT and S1P 1 (S5A) EAE mice (day 8 post-immunization) treated in vitro with recombinant IL-6 (IL-6) or anti-IL-6 (α-IL-6) (20ng/ml, respectively). ( d ) Quantification of normalized p-STAT3 expression from ( c ). IL-17 expression in the culture supernatants of splenocyte cultures from MOG 35–55 -immunized S1P 1 (S5A) mice treated in vitro with Stattic (STAT3 inhibitor) (0–9 μM) ( e ) or Jak inhibitor (0–5nM)( f ). ** p <0.01, * p <0.05 by Students t -test. Experiments were performed 3–5 times with n =3–5 mice.

Article Snippet: WT C57BL/6J or S1P 1 (S5A) mice (female, 8–9 weeks old) were immunized with CFA and MOG 35–55, splenocytes and lymph node cells were harvested on day 9 post-immunization and expanded in vitro with MOG 10 μg/ml and IL-12 (20 ng/ml)(R&D Systems) for 72 h . Cells were then harvested, washed once with pre-warmed PBS, counted and injected into 5–6 weeks old Rag1 −/− naïve recipient mice (1×10 7 cells per mouse) intraperitoneally and followed clinically up to at least day 30.

Techniques: Expressing, Cell Culture, Irradiation, Enzyme-linked Immunosorbent Assay, Ex Vivo, Western Blot, In Vitro, Recombinant