Journal: European journal of immunology

Article Title: A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice

doi: 10.1002/eji.201546095

Figure Lengend Snippet: IL-23p19 and IL-27/IL-35Ebi3 can form a heterodimer, termed IL-39. (A) Detection and characterization of human IL-39 by immunoprecipitation (IP) and Western blot (WB) analyses under reducing conditions. (B) Schematic of the cDNA constructs used to genetically engineer mouse p19 and Ebi3 proteins. hPGK: human phosphoglycerate kinase 1 promoter. (C) Detection and characterization of mouse IL-39 recombinant proteins by IP and Western blot analyses under reducing conditions. (D) Coomassie Blue gel of the recombinant mouse IL-39 characterized on nonreducing polyacrylamide gels. Arrow indicates recombinant IL-39. (A, C, and D) Blots are representative of three independent experiments.

Article Snippet: A 5 ng/mL human IL-27/IL-35 Ebi3 subunit (Origene) and 5 ng/mL human IL-23 p19 subunit (Origene) were mixed in PBS.

Techniques: Immunoprecipitation, Western Blot, Construct, Recombinant

Journal: European journal of immunology

Article Title: A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice

doi: 10.1002/eji.201546095

Figure Lengend Snippet: In vitro activated B cells expressed IL-39. (A, B) Primary B cells were sorted from 8-weeks-old female C57BL/6 mice by B220 microbeads, stimulated for 48 h with LPS, and analyzed by FACS. (A) The percentages of IL-39-expressing B cells and (B) statistical analysis of the percentage of IL-12 family cytokine subunits are shown. Isotype was used as the staining control. (C) The concentration of IL-39 in the cultured supernatant from LPS-stimulated B cells was measured by sandwich ELISA by using anti-p19 and Ebi3 antibody as coated and detected antibody, respectively. (B and C) Data are shown as mean + SEM (n = 8) from one experiment representative of three other similar experiments. (D) Detection of mouse natural IL-39 proteins in the cultured supernatant on days 3 after B cells were stimulated with LPS by IP and Western blot analyses under reducing conditions. Blots are representative of three independent experiments *p < 0.05, **p < 0.01, ***p < 0.001 (two tailed Student’s t-test).

Article Snippet: A 5 ng/mL human IL-27/IL-35 Ebi3 subunit (Origene) and 5 ng/mL human IL-23 p19 subunit (Origene) were mixed in PBS.

Techniques: In Vitro, Expressing, Staining, Control, Concentration Assay, Cell Culture, Sandwich ELISA, Western Blot, Two Tailed Test

Journal: European journal of immunology

Article Title: A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice

doi: 10.1002/eji.201546095

Figure Lengend Snippet: IL-39 induces inflammation in lupus-like Mice. (A) GL7+ B cells were sorted from 8-month-old female lupus-like MRL/lpr mice were sorted by FACS and infected with control, IL-39 subunits p19 or Ebi3-specific shRNA. On day 1 after infection, p19 and Ebi3 mRNA expression were analyzed by qPCR. (B–D) A 5 × 106 control, p19 or Ebi3-specific shRNA-infected GL7+ B220+ B cells per mouse were i.v. injected into 8-week-old female lupus-like MRL/lpr mice (six mice per group). Eight-week-old female lupus-like MRL/lpr mice into which either no cells, or transferred with control shRNA-infected GL7+ B cells, were used as no-cells transfer (None) and shRNA (Control) controls respectively. (B) On day 14 after cell transfer, spleens were harvested and photographed (representative of six spleens per experiments from three independent experiments). (C) Proteinuria was measured on day 0 and day 4 after cell transfer. (D) Absolute numbers of B220+ B and GL7+ B per spleen on days 7 after cell transfer. (A, C, and D) Data are shown as mean + SEM (n = 6) from one experiment representative of two other similar experiments. *p < 0.05, **p < 0.01 (two tailed Student’s t-test).

Article Snippet: A 5 ng/mL human IL-27/IL-35 Ebi3 subunit (Origene) and 5 ng/mL human IL-23 p19 subunit (Origene) were mixed in PBS.

Techniques: Infection, Control, shRNA, Expressing, Injection, Two Tailed Test

Journal: European journal of immunology

Article Title: A novel IL-23p19/Ebi3 (IL-39) cytokine mediates inflammation in Lupus-like mice

doi: 10.1002/eji.201546095

Figure Lengend Snippet: IL-39 signals through IL-23R and gp130 receptor subunits and activates STAT1 and STAT3 pathways. (A) Primary B cells were sorted from 8-week-old female C57BL/6 mice by B220 microbeads, stimulated for 48 h with LPS, washed, and starved for 2 h in serum- free medium (0.5% BSA), followed by stimulation for 30 min with medium, p19, Ebi3, or IL-39. STAT activation was analyzed by Western blotting. Blots are representative of four independent experiments. (B) Primary B cells were sorted from 8-week-old female C57BL/6 mice by B220 microbeads, stimulated for 24 h with LPS in the presence of medium p19, Ebi3, or IL-39. IL-39 subunits (p19 and Ebi3) mRNA was determined using qPCR assay. Data are shown as mean + SEM (n = 8) from one experiment representative of two other similar experiments. (C) Control or IL-23R, IL-27Ra, gp130-specific shRNA-infected B cells described in Supporting Information Fig. 3C were stimulated for 2 days with LPS, washed, and starved for 2 h in serum-free medium (0.5% BSA), followed by stimulation for 30 min with IL-39. Cell lysates were separated by 8% SDS-PAGE and immunoblotted for phospho-Tyr701-STAT1 (pSTAT1) and total STAT1 (upper and left panel) or phosphor-Tyr705-STAT3(pSTAT3)andtotalSTAT3(upper and right panel). Band intensities of pSTAT1 and STAT1 or pSTAT3 and STAT3 were quantified by ImageProPlus 5.0 software. The density ratios of phosphorylated to total protein compared to control shRNA-infected group are shown as mean ± SEM (n = 3) of three independent experiments (lower panel). *p < 0.05, **p < 0.01 (two tailed Student’s t-test).

Article Snippet: A 5 ng/mL human IL-27/IL-35 Ebi3 subunit (Origene) and 5 ng/mL human IL-23 p19 subunit (Origene) were mixed in PBS.

Techniques: Activation Assay, Western Blot, Control, shRNA, Infection, SDS Page, Software, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-12 p40 monomer is different from other IL-12 family members to selectively inhibit IL-12Rβ1 internalization and suppress EAE

doi: 10.1073/pnas.2000653117

Figure Lengend Snippet: Level of p40 in serum of EAE mice and MS patients. Female SJL/J mice were induced EAE by adoptive transfer of MBP-primed T cells. At the acute phase (14 dpt), levels of IL-12 (A), IL-23 (B), p402 (C), and p40 (D) were measured in serum by sandwich ELISA. Results are mean ± SEM of six mice per group. Level of p40 was measured in homogenates of the spleen, cerebellum and spinal cord at acute (14 dpt) and remission (22 dpt) phases of EAE (E). Results are mean ± SEM of six mice per group. aP < 0.01 vs. control; bP < 0.001 vs. EAE acute phase spleen; cP < 0.001 vs. EAE acute phase cerebellum; dP < 0.001 vs. EAE acute phase spinal cord. Serum of MS patients with active disease (n = 10) and age-matched healthy controls (n = 10) was analyzed for IL-12 (F), p402 (G), and p40 (H) by sandwich ELISA. *P < 0.05; ***P < 0.001.

Article Snippet: While recombinant mouse p40 monomer (p40) was obtained from BD Bioscience, recombinant human p40 was obtained from R&D Systems.

Techniques: Adoptive Transfer Assay, Sandwich ELISA

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-12 p40 monomer is different from other IL-12 family members to selectively inhibit IL-12Rβ1 internalization and suppress EAE

doi: 10.1073/pnas.2000653117

Figure Lengend Snippet: Levels of p40, p40 2 , and IL-12 in serum of MS patients and control subjects

Article Snippet: While recombinant mouse p40 monomer (p40) was obtained from BD Bioscience, recombinant human p40 was obtained from R&D Systems.

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-12 p40 monomer is different from other IL-12 family members to selectively inhibit IL-12Rβ1 internalization and suppress EAE

doi: 10.1073/pnas.2000653117

Figure Lengend Snippet: Treatment of EAE by recombinant p40. (A) Mouse p40 (BD Bioscience) was run through native PAGE followed by Coomassie blue staining. (B) Adoptively transferred EAE mice were treated with different doses of p40 once a week via i.p. injection starting from 0 dpt. Mice were examined for clinical symptoms for the next 30 d. Data are expressed as the mean ± SEM of six mice per group. *P < 0.05 vs. EAE+p40 (25 ng per mouse); ***P < 0.001 vs. EAE+p40 (200 ng per mouse). Repeated measures one-way ANOVA was calculated with treatment as a single factor, and the outcome was summarized as F4,160 = 14.8 (>Fc = 4.32). (C) Adoptively transferred EAE mice were treated with recombinant mouse p40 and heat-inactivated p40 (200 ng per mouse) weekly via i.p. injection starting from 8 dpt (the onset of acute phase). Data are expressed as the mean ± SEM of six mice per group. ***P < 0.001 vs. EAE+p40. (D) Adoptively transferred EAE mice received one i.p. injection of p40 mAb a3-3a (100 μg per mouse) on 8 dpt. Another group of mice also received the same amount of control hamster IgG. Mice were examined for clinical symptoms daily until 30 dpt. Data are expressed as the mean ± SEM of six mice per group. *P < 0.05 vs. EAE+p40. (E) Adoptively transferred EAE mice were treated with p40 (200 ng per mouse) weekly starting from 19 dpt (the onset of relapsing phase). Data are expressed as the mean ± SEM of six mice per group. **P < 0.01 vs. EAE+p40. (F) MOG-induced active EAE mice were treated with p40 (200 ng per mouse) weekly starting from 10 dpt (the onset of acute phase). Data are expressed as the mean ± SEM of six mice per group. ***P < 0.001 vs. EAE+p40. (G) Th17 cell-induced active EAE mice were treated with p40 (200 ng per mouse) weekly starting from 8 dpt. Data are expressed as the mean ± SEM of six mice per group. ***P < 0.001 vs. EAE+p40. (H) PLP139-151–induced chronic RR-EAE mice were treated with p40 (200 ng per mouse) weekly starting from 9 dpt. Data are expressed as the mean ± SEM of six mice per group. *P < 0.05 vs. EAE+p40. (I) Adoptively transferred EAE mice were treated with recombinant mouse IL-12, IL-23, or p402 (200 ng per mouse) once on 8 dpt. Data are expressed as the mean ± SEM of six mice per group. *P < 0.05. (J) Adoptively transferred EAE mice were treated with recombinant human p40 (200 ng per mouse) weekly starting from 8 dpt. Data are expressed as the mean ± SEM of six mice per group. ***P < 0.001 vs. EAE+p40.

Article Snippet: While recombinant mouse p40 monomer (p40) was obtained from BD Bioscience, recombinant human p40 was obtained from R&D Systems.

Techniques: Recombinant, Clear Native PAGE, Staining, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-12 p40 monomer is different from other IL-12 family members to selectively inhibit IL-12Rβ1 internalization and suppress EAE

doi: 10.1073/pnas.2000653117

Figure Lengend Snippet: Effect of recombinant p40 on the CNS infiltration of mononuclear cells and demyelination in EAE mice. (A) Cerebellar sections isolated from normal, EAE (14 dpt) and p40-, p402-, or IL-12–treated EAE (14 dpt receiving these cytokines from 8 dpt) mice were stained with hematoxylin/eosin. Digital images were collected under bright field setting using a ×40 objective. Infiltration (B) and cuffed vessel (C) in cerebellar sections were represented quantitatively by using a scale as described in SI Appendix, SI Materials and Methods. Data are expressed as the mean ± SEM of four mice per group. (D) Mononuclear cells (MNCs) isolated from the cerebellum of EAE and EAE+p40 mice on 14 dpt were analyzed by FACS in an LSRFortessa analyzer (BD Biosciences). MNCs were gated, and percentages of CD4+ (E) and CD8+ (F) T cells in that gate were quantitatively analyzed. Data represent mean ± SEM of four mice per group. Longitudinal (G) and transverse (H) sections of the spinal cord and coronal sections of the cerebellum (I) isolated from normal, RR-EAE (14 dpt), and p40-, p402-, or IL-12–treated RR-EAE (14 dpt receiving these cytokines from 8 dpt) mice were stained with LFB. Digital images were collected under bright field setting using a ×40 objective. Demyelination in the spinal cord (J) and cerebellum (K) was represented quantitatively by using a scale as described in SI Appendix, SI Materials and Methods. On 8 dpt, mice were treated with 200 ng per mouse p40, p402, or IL-12 via i.p. injection. Data are expressed as the mean ± SEM of four mice per group. ***P < 0.001.

Article Snippet: While recombinant mouse p40 monomer (p40) was obtained from BD Bioscience, recombinant human p40 was obtained from R&D Systems.

Techniques: Recombinant, Isolation, Staining, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-12 p40 monomer is different from other IL-12 family members to selectively inhibit IL-12Rβ1 internalization and suppress EAE

doi: 10.1073/pnas.2000653117

Figure Lengend Snippet: The recombinant p40 protects mice from CIA. (A) CIA was induced in male DBA/1J mice by bovine type II collagen immunization, and, from 29 dpi, mice were treated with p40 (200 ng per mouse) weekly via i.p. injection. Mice (n = 6 per group in two independent experiments) were scored daily. (B) On 60 dpi, images of swollen paws were taken. (C) Paw thickness was monitored in six mice per group in two different experiments. Repeated measures one-way ANOVA was calculated with treatment as a single factor, and the outcome was summarized as F2,36 = 50.77 (>Fc = 4.68). General motor activities were monitored by the Ethovision System (D, heat map images representing overall motor activities; E, distance traveled; F, velocity; G, center movement), grip strength (H), and rotorod (I). Foot print analysis (J, stride length; K, toe spread; L, print length; M, sway length) was also performed. Levels of TNFα (N), IL-1β (O), and nitrite (P) were also monitored in serum. Six mice (n = 6 per group) were used in two independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-sample t tests.

Article Snippet: While recombinant mouse p40 monomer (p40) was obtained from BD Bioscience, recombinant human p40 was obtained from R&D Systems.

Techniques: Recombinant, Injection

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-12 p40 monomer is different from other IL-12 family members to selectively inhibit IL-12Rβ1 internalization and suppress EAE

doi: 10.1073/pnas.2000653117

Figure Lengend Snippet: The p40 treatment retains the surface expression of IL-12Rβ1, but neither IL-12Rβ2 nor IL-23R, in MBP-primed T cells. (A) Splenocytes isolated from MBP-immunized donor mice were restimulated with MBP in the presence or absence of p40, IL-12, p40+IL-12, p402, p402+p40, IL-23, and IL-23+p40 for 4 h followed by FACS analysis of nonadherent cells in an LSRFortessa analyzer (BD Biosciences) for IL-12Rβ1 and CD4. Where cells were treated with the combination of cytokines, p40 was used 30 min prior to p402, IL-12, or IL-23. (B) Under similar treatment conditions (p40, IL-12, p40+IL-12), the surface expression of IL-12Rβ2 was monitored by FACS. (C) Under similar treatment conditions (p40, IL-23, p40+IL-23), the surface expression of IL-23 was monitored by FACS. The MFI of IL-12Rβ1 (D), IL-12Rβ2 (E), and IL-23R (F) in the CD4+ population was calculated by using the CellQuest software. Data are mean ± SD of four different experiments. ***P < 0.001. ns, not significant; PE, phycoerythrin; FITC, fluorescein isothiocyanate.

Article Snippet: While recombinant mouse p40 monomer (p40) was obtained from BD Bioscience, recombinant human p40 was obtained from R&D Systems.

Techniques: Expressing, Isolation, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-12 p40 monomer is different from other IL-12 family members to selectively inhibit IL-12Rβ1 internalization and suppress EAE

doi: 10.1073/pnas.2000653117

Figure Lengend Snippet: The p40 inhibits the phosphorylation of STAT4 in MBP-primed T cells. Splenocytes isolated from MBP-immunized donor mice were restimulated with MBP (A–C) in the presence of IL-12 (D–F) and p40+IL-12 (G–I) for different time periods, followed by Western blot of STAT4 and pSTAT4 in nonadherent splenocytes. Cells were treated with p40 30 min before IL-12. Actin was used as a loading control. Bands were scanned, and values of pSTAT4/STAT4 (B, MBP; E, MBP+IL-12; H, MBP+IL-12+p40) and STAT4/β-actin (C, MBP; F, MBP+IL-12; I, MBP+IL-12+p40) are presented as relative to control. Data are expressed as the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Article Snippet: While recombinant mouse p40 monomer (p40) was obtained from BD Bioscience, recombinant human p40 was obtained from R&D Systems.

Techniques: Isolation, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-12 p40 monomer is different from other IL-12 family members to selectively inhibit IL-12Rβ1 internalization and suppress EAE

doi: 10.1073/pnas.2000653117

Figure Lengend Snippet: Enrichment of Tregs by p40. Splenocytes isolated from MBP-immunized donor mice were restimulated with MBP in the presence or absence of p40 (10 ng/mL), IL-23 (10 ng/mL), and p40+IL-23 for 48 h, followed by FACS analysis of nonadherent cells in an LSRFortessa analyzer (BD Biosciences) for CD4 and Foxp3 (A). The MFI of Foxp3 (B) in the CD4+ population was calculated by using the CellQuest software. Results are mean ± SD of three different experiments. **P < 0.01 and ***P < 0.001 by two-sample t tests. Splenocytes isolated from MBP-immunized donor mice were treated with p40 (30 ng/mL), followed by restimulation with MBP in the presence of either Th1 (C and D) or Th17 (E and F) polarization (Pol) as mentioned in SI Appendix, SI Materials and Methods. After 48 h, cells were analyzed by FACS for CD4 and Foxp3 (C, Th1 polarization; E, Th17 polarization). The MFI of Foxp3 (D, Th1 polarization; F, Th17 polarization) in the CD4+ population was calculated by using the CellQuest software. Results are mean ± SD of three different experiments. **P < 0.01; ***P < 0.001 by two-sample t tests.

Article Snippet: While recombinant mouse p40 monomer (p40) was obtained from BD Bioscience, recombinant human p40 was obtained from R&D Systems.

Techniques: Isolation, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: IL-12 p40 monomer is different from other IL-12 family members to selectively inhibit IL-12Rβ1 internalization and suppress EAE

doi: 10.1073/pnas.2000653117

Figure Lengend Snippet: Involvement of IL-12Rβ1 and/or IL-12Rβ2 in p40-mediated protection of EAE. (A) EAE was induced in IL-12Rβ1−/− (Rβ1−/−) and IL-12Rβ2−/− (Rβ2−/−) mice by MOG immunization. We did not observe EAE symptoms in IL-12Rβ1−/− mice. From 8 dpi, IL-12Rβ2−/− mice were treated with p40 (200 ng per mouse) once a week via i.p. injection. Mice were examined for clinical symptoms until 34 dpi. Data are expressed as the mean ± SEM of six mice per group. ***P < 0.001 vs. EAE+p40. On 20 dpi, levels of IFNγ (B) and IL-17 (C) were monitored in serum by ELISA. Results are mean ± SEM of four mice per group. Splenocytes were analyzed by FACS in an LSRFortessa analyzer (BD Biosciences) for CD4 and IFNγ and CD4 and IL-17. The MFI of IFNγ (D) and IL-17 (E) in the CD4+ population was calculated by using the CellQuest software. ***P < 0.001 by two-sample t tests. Rβ1−/− mice were bred with Rβ2−/− mice to generate Rβ1+/−/Rβ2−/− mice. Genotyping data are presented (F). Whole spleens are shown for all different groups (G). There were no significant differences in spleen weight (H) and total body weight (I) among different groups of mice (8 wk old). (J) EAE was induced in Rβ1+/−/Rβ2−/− mice by MOG immunization, followed by treatment with p40 (200 ng per mouse) once a week via i.p. injection from 8 dpi. Mice were scored until 34 dpi. Data are expressed as the mean ± SEM of six mice per group. *P < 0.05 vs. p40 treatment.

Article Snippet: While recombinant mouse p40 monomer (p40) was obtained from BD Bioscience, recombinant human p40 was obtained from R&D Systems.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Software