carrier free recombinant mouse il  (R&D Systems)

Journal: Mucosal immunology

Article Title: Regulation of IL-12p40 by HIF controls Th1/Th17 responses to prevent mucosal inflammation.

doi: 10.1038/mi.2016.135

Figure Lengend Snippet: Figure 2 Analysis of PHDi-driven Th1 and Th17 responses in the colonic mucosa and MLNs of mice with chemically-induced colitis. (a) Colon tissue- cytokine analysis of IFN-g, IL-12p70, IL-23 and IL-12p40 protein levels from healthy or DSS-treated animals treated with PHDi (5 mg kg 1 AKB-4924) or vehicle (veh) (cyclodextrin). (b) RT-PCR analysis of il12a, il12b and il23a expression in whole colon tissue of DSS mice receiving vehicle or PHDi, compared with healthy animal receiving vehicle (n ¼ 6–11). Flow cytometric analysis of intracellular cytokine production from LPL and MLNs in PHDi treated (AKB-4924 5mg/kg) or vehicle (veh) mice on day 10 of DSS colitis (n ¼ 18). (c) Relative expression of il12a, il23a and il12b mRNA levels in the colon of control and TNBS animals treated with vehicle of PHDi (d) Representative dot plots of IFN-g and IL-17 producing CD3 þCD4 þ T cells from LPL. (e) LPL þ T cells producing IFN-g or IL-17, expressed as total number and % live CD3 þCD4 population. (f) Representative dot plots of MLN CD3 þCD4 þ T cells producing IFN-g or IL-17. (g) MLN CD3 þCD4 þ T cells producing IFN-g or IL-17 expressed as % live CD3 þCD4 þ cells. (n ¼ 6, 5mg/kg AKB-4924 every 48 hours). Values are presented as mean ±s.e.m. (a, b, g) one-way ANOVA, (d, f) student’s t-test, ns ¼ not significant, *Po0.05, **Po0.01. ANOVA, analysis of variance; DSS, dextran sulfate sodium; LPL, lamina propria lymphocyte; TNBS, 2,4,6-trinitrobenzenesulfonic acid; MLN, mesenteric lymph node; RT-PCR, real-time PCR.

Article Snippet: Recombinant IL-12p40 homodimer (#499-ML-025) and Mouse AntiIL-23 p19 Antibody (#AF1619) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Real-time Polymerase Chain Reaction

Journal: Mucosal immunology

Article Title: Regulation of IL-12p40 by HIF controls Th1/Th17 responses to prevent mucosal inflammation.

doi: 10.1038/mi.2016.135

Figure Lengend Snippet: Figure 3 Regulation of IL-12 and IL-23 subunit expression by PHDi in vitro. RAW 246.7 macrophages were cultured in vitro with either vehicle or PHDi (AKB-4924 5 mM) for 10 hours, either in (a) medium alone or (b) with LPS (100 ng ml 1). RT-PCR was used to determine the expression of il12b, il12a, or il23a in these cultures. Supernatants were collected from LPS-stimulated RAW 246.7 macrophages and (c) examined by immunoblot and (d) densitometry for IL-12p40-containing proteins under denaturing and non-denaturing conditions. Densitometry analysis was performed with ImageJ to examine IL-12p40-containing. Data expressed as mean±s.e.m. (a,b) or fold change over vehicle±s.e.m. (c) (n ¼ 6). ***Po0.005. (a,b) two-way ANOVA, (c) students t-test, ns, not significant, *Po0.05. ANOVA, analysis of variance; LPS, lipopolysaccharide; RT-PCR, real-time PCR.

Article Snippet: Recombinant IL-12p40 homodimer (#499-ML-025) and Mouse AntiIL-23 p19 Antibody (#AF1619) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, In Vitro, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot, Real-time Polymerase Chain Reaction

Journal: Mucosal immunology

Article Title: Regulation of IL-12p40 by HIF controls Th1/Th17 responses to prevent mucosal inflammation.

doi: 10.1038/mi.2016.135

Figure Lengend Snippet: Figure 4 Analysis of hypoxia-responsive elements in the human and murine il12b promoter. Immunoblot analysis of IL-12p40 homodimer secretion from human (a) peripheral blood monocytes or (b) lamina propria monocyte cells stimulated with LPS in the presence of absence of cyclodextran vehicle (Veh), PHDi (AKB-4924, 5 mM) or hypoxia (Hx), with (c) densitometry analysis representing the ratio of IL-12p40 homodimer to IL-12p70. (d) Schematic representation of hypoxia-responsive elements (HRE) in the il12b promoter in mouse and human and (e) subsequent analysis of the influence of hypoxia or PHDi-treatment on iL12b promoter activity in U937 cells transfected with an il12b-luciferase promoter reporter vector. (f) In silico analysis of HBS and HAS in the murine and human il12b promoter with predicted similarity to HRE function (Matrix similarity, max ¼ 1). (g) Site-directed mutagenesis of HIF- binding sites in the human (U937-transfected cells) and murine (RAW 264.7 transfected cells) il12b promoter and subsequent response to PHDi- treatment or hypoxia, in comparison to normal oxygen tensions (normoxia). For reporter transfection studies, renilla was employed as a co-transfection control and data expressed as mean RLU ratio il12b:renilla±s.e.m. (n ¼ 4). For densitometry analysis, data expressed as mean±s.e.m. (c, g) one-way ANOVA, (e) student’s t-test, wPo0.05, *Po0.05. ANOVA, analysis of variance; HAS, HIF ancillary sites; HIF ancillary sites, HIB; HIF, hypoxia-inducible factor-1a; LPS, lipopolysaccharide; RLU, relative luminescence units.

Article Snippet: Recombinant IL-12p40 homodimer (#499-ML-025) and Mouse AntiIL-23 p19 Antibody (#AF1619) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Activity Assay, Transfection, Luciferase, Plasmid Preparation, In Silico, Mutagenesis, Binding Assay, Comparison, Cotransfection, Control

Journal: Mucosal immunology

Article Title: Regulation of IL-12p40 by HIF controls Th1/Th17 responses to prevent mucosal inflammation.

doi: 10.1038/mi.2016.135

Figure Lengend Snippet: Figure 5 Influence of PHDi treatment on colitis-associated pathology Th responses in the absence of IL-12p40. Groups were treated at 48 hour intervals with PHDi (AKB-4924, 5 mg kg 1) or vehicle (veh, cyclodextrin). (a) Disease progress was assessed by weight change (%) from initial weight and (b) mucosal thickening of the colon expressed as weight (mg)/length (cm). Upon sacrifice, pathology was assessed by (c) blinded histological inflammation and colitis score. Panel (d) shows representative H&E-stained histological images of colons from naive IL-12p40 / (Naive KO) animals and DSS IL-12p40 / (DSS KO) animals with and without PHDi treatment. Flow cytometric analysis was performed to examine intracellular cytokine production from LPL restimulated in vitro in DSS WT or IL-12p40 / mice receiving PHDi (AKB-4924 5 mg kg 1) or vehicle treatment (Veh). (e) Representative dot plots of LPL CD3 þCD4 þ T cells producing IFN-g, or IL-17 in DSS wildtype or IL-12p40 / mice. (f) LPL CD3 þCD4 þ T cells producing IFN-g or IL-17, expressed as total number and % live (g) Analysis of IL-1b and IL-6 mRNA transcript and protein levels in the colonic mucosa of control and DSS willdtype or IL-12p40 / mice. Data expressed as mean ±s.e.m., (n ¼ 6-9 per group). (a) Two-way ANOVA, (b, g) student’s t-test, (d, f) one-way ANOVA, *Po0.05, **Po0.01. Scale bar,100 mm. ANOVA, analysis of variance; DSS, dextran sulfate sodium; H&E, hematoxylin and eosin; KO, knockout; WT, wild type.

Article Snippet: Recombinant IL-12p40 homodimer (#499-ML-025) and Mouse AntiIL-23 p19 Antibody (#AF1619) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Staining, In Vitro, Control, Knock-Out

Journal: Mucosal immunology

Article Title: Regulation of IL-12p40 by HIF controls Th1/Th17 responses to prevent mucosal inflammation.

doi: 10.1038/mi.2016.135

Figure Lengend Snippet: Figure 7 The influence of recombinant IL-12p40 homodimer treatment on the development of DSS colitis associated pathology. Groups received daily i.p. treatments with 10 ng of recombinant IL-12p40 homodimer (rIL-12p40h), PHDi or vehicle (Veh) as indicated. (a) Weight change (%) from initial weight in the wildtype (WT) or IL-12p40 / mice receiving DSS and treated with vehicle, PHDi or rIL-12p40h (* denotes DSS WT þ PHDi and w denotes DSS WT þ rIL-12p40h compared against DSS WT þ Veh). (b) Pathological mucosal thickening of the colon after DSS treatment, expressed as weight (mg)/ length (cm) and DSS-induced colon-shortening. (c) Percentage of LPL CD3 þCD4 þ T cells producing IFN-g in the colon of DSS WT or IL-12p40 / mice treated with vehicle or rIL-12p40h. (d) il12b mRNA expression in tissue isolated from the colonic mucosa of DSS wild-type mice treated with vehicle or rIL-12p40 homodimer. (e) Representative H&E colon sections and inflammation and epithelial score for animals receiving anti-IL-23p19 (20 mg kg 1, i.p) or control (Rat IgG2a, 20 mg/Kg, i.p) daily from day 0 and treated with vehicle, PHDi or rIL-12p40h from day 5 as indicated. (f) Weight change (%) from initial weight in DSS-treated animals receiving anti-IL-23p19 (daily 20 mg/kg 1, i.p) or control (Rat IgG2a, 20 mg kg 1, i.p) and treated with vehicle, PHDi or rIL-12p40h as indicated. (g) Weight change (%) from initial weight in TNBS models of colitis treated with vehicle, PHDi or rIL-12p40h. (h) Pathological mucosal thickening of the colon expressed as weight (mg)/length (cm) in the TNBS model of colitis. Data expressed as mean±s.e.m., (a, e and f) two-way ANOVA (n ¼ 6 per group), (b–d) one-way ANOVA (n ¼ 6 per group) (g, h) student’s t-test, (n ¼ 6 per group) * and wPo0.05 ** and ww Po0.01, ***Po0.005. Scale bar, 100 mm. ANOVA, analysis of variance; DSS, dextran sulfate sodium; H&E, hematoxylin and eosin; TNBS, 2,4,6-trinitrobenzenesulfonic acid; WT, wild-type.

Article Snippet: Recombinant IL-12p40 homodimer (#499-ML-025) and Mouse AntiIL-23 p19 Antibody (#AF1619) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Recombinant, Expressing, Isolation, Control

Journal: Mucosal immunology

Article Title: Regulation of IL-12p40 by HIF controls Th1/Th17 responses to prevent mucosal inflammation.

doi: 10.1038/mi.2016.135

Figure Lengend Snippet: Figure 8 Potential role for IL-12p40 homodimer as an ‘‘inflammatory brake’’ during hypoxic inflammation. Activation of HIF via hypoxia or therapeutic PHDi treatment leads to a selective induction of IL-12p40 and secretion of IL-12p40 homodimer by myeloids cells, which suppress the differentiation of naive T helper cells into Th1cells or the stabilization of Th17 cells, limiting the progression of Th1/th17 inflammation. PHDi: prolyl-hydroxylase inhibitor, Hx: hypoxia Th0: naive T helper cell. HIF, hypoxia-inducible factor-1a.

Article Snippet: Recombinant IL-12p40 homodimer (#499-ML-025) and Mouse AntiIL-23 p19 Antibody (#AF1619) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay

Journal: JCI Insight

Article Title: Allograft dendritic cell p40 homodimers activate donor-reactive memory CD8 + T cells

doi: 10.1172/jci.insight.96940

Figure Lengend Snippet: (A) Groups of A/J mice (n = 4–8/group) received cardiac allografts subjected to 0.5 or 8 hours of CIS from either WT C57BL/6 (WTB6) or B6.IL12p40–/– donors. The indicated recipients of WT allografts were treated with 200 μg anti–IL-12 p40 mAb i.p. on days 0 and 1. All recipients were injected with 100 μg BrdU i.p. on days 0 and 1 after transplant. The following day, allografts were harvested and digested, and cell aliquots were stained with antibody and analyzed by flow cytometry to assess total numbers and proliferation of infiltrating memory CD4+ and CD8+ T cells. *P < 0.05; **P < 0.01, as determined by the Mann-Whitney nonparametric test. (B) Groups of A/J mice (n = 4/group) received cardiac allografts subjected to 0.5 or 8 hours of CIS from WT C57BL/6 or B6.IL12p35–/– donors. All recipients were injected with 100 μg BrdU i.p. on days 0 and 1. On day 2 after transplant, allografts were harvested and digested, and cell aliquots were stained with antibody and analyzed by flow cytometry to assess total numbers and proliferation of infiltrating memory CD4+ and CD8+ T cells. *P < 0.05 as determined by the Mann-Whitney nonparametric test. (C) Groups of C57BL/6 mice (n = 5–6/group) received C57BL/6 cardiac isografts or A/J cardiac allografts subjected to 0.5 or 8 hours of CIS. Grafts were harvested on day 2 after transplant, total RNA was isolated, and expression of the indicated cytokine mRNA was tested by qPCR. Results shown indicate relative expression vs. expression in naive hearts of A/J mice. **P < 0.01, as determined by the Mann-Whitney nonparametric test.

Article Snippet: Quantification of IL-12 heterodimer and p40 homodimer protein was performed on heart iso- and allograft lysates, and serum was collected at 48 hours after transplant using Mouse IL-12 p70 and Mouse p40 Quantikine ELISA Kits, respectively, from R&D Systems.

Techniques: Injection, Staining, Flow Cytometry, MANN-WHITNEY, Isolation, Expressing

Journal: JCI Insight

Article Title: Allograft dendritic cell p40 homodimers activate donor-reactive memory CD8 + T cells

doi: 10.1172/jci.insight.96940

Figure Lengend Snippet: (A) Groups of C57BL/6 mice (n = 5/group) received A/J cardiac allografts or C57BL/6 isografts subjected to 0.5 or 8 hours of CIS. The indicated recipients of 8 hours of CIS allografts were treated with 200 μg control rat IgG or anti-CD4 mAb on days –3, –2, and –1 prior to transplant. On day 2 after transplant, serum and graft lysates were prepared and quantities of p40 homodimers and IL-12 p70 heterodimers were tested by ELISA. *P < 0.05, as determined by the Mann-Whitney nonparametric test. (B) Groups of C57BL/6 mice (n = 5/group) received A/J cardiac allografts subjected to 0.5 or 8 hours of CIS and the indicated recipients were injected with 200 μg recombinant p40 homodimers i.p. on days 0 and 1. All recipients were injected with 100 μg BrdU i.p. on days 0 and 1 after transplant; the next day, allografts were harvested and digested, and cell aliquots were stained with antibody and analyzed by flow cytometry to assess total numbers and numbers of proliferating memory CD8+ T cells infiltrating the grafts. *P < 0.05; **P < 0.01, as determined by the Mann-Whitney nonparametric test. (C) Groups of C57BL/6 mice (n = 4–7/group) received A/J cardiac allografts subjected to 0.5 or 8 hours of CIS, and on days 0 and 1, were treated with 200 μg control rat IgG or 250 μg CTLA-4Ig i.p. with or without 200 μg recombinant p40 homodimers i.p. Allograft survival was monitored by daily abdominal palpation and rejection confirmed by laparotomy. ***P < 0.001 vs. all other groups, as determined using the Log-rank/Mantel-Cox test.

Article Snippet: Quantification of IL-12 heterodimer and p40 homodimer protein was performed on heart iso- and allograft lysates, and serum was collected at 48 hours after transplant using Mouse IL-12 p70 and Mouse p40 Quantikine ELISA Kits, respectively, from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Injection, Recombinant, Staining, Flow Cytometry