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