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Supplementary Materials Deficiency of the IL-23/IL-17 axis accelerates renal injury in mice with DOCA+Ang II induced hypertension Christian F. Krebs 1*,

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Presentation on theme: "Supplementary Materials Deficiency of the IL-23/IL-17 axis accelerates renal injury in mice with DOCA+Ang II induced hypertension Christian F. Krebs 1*,"— Presentation transcript:

1 Supplementary Materials Deficiency of the IL-23/IL-17 axis accelerates renal injury in mice with DOCA+Ang II induced hypertension Christian F. Krebs 1*, Sascha Lange 1*, Gianina Niemann 1, Alva Rosendahl 1, Alexander Lehners 1, Catherine Meyer-Schwesinger 1, Rolf A.K. Stahl 1, Ralf A. Benndorf 2, Joachim Velden 3, Hans Joachim Paust 1, Ulf Panzer 1, Heimo Ehmke 2 and Ulrich O. Wenzel 1 1 Department of Medicine and 2 Department of Physiology, University Hospital Hamburg- Eppendorf, Hamburg; 2 Institute of Anatomy and Cell Biology, University of Wuerzburg; 3 Department of Nephropathology, University Hospital Erlangen, Germany Correspondence to: Ulrich Wenzel III. Medizinische Klinik University Hospital Hamburg-Eppendorf Martinistr. 52; Hamburg, Germany Tel: Fax:

2 Methods Mice and experimental groups IL-17A -/-, IL-23p19 -/- and matched wildtype C57BL/6J mice were used as described by us week-old IL-17A -/- mice and wildtype mice were assigned into 3 groups: normotensive wildtype, wildtype mice with DOCA+Ang II and IL-17 -/- mice with DOCA+Ang II. In a second experiment the same protocol was used with the IL-23p19 -/- mice. 1 DOCA+Ang II induced hypertension Hypertension was induced using our recently developed model of DOCA+Ang II-induced hypertension 2, 3. At day 0 mice were uninephrectomized. At day 14 a 50 mg DOCA pellet (Deoxycorticosteron Acetate; Innovative Research, USA) was implanted and mice received 0.9% NaCl in the drinking water. At day 21 an osmotic minipump (Alzet 1002, Cupertino, USA) delivering 1.2 ng Ang II (Sigma, USA) per minute and gram body weight was subcutaneously implanted. Mice were sacrificed at day 4 (initial injury) or 14 after start of Ang II infusion. More details of Methods used for this work have been described before by us 1, 2, 4, 5 and can be found in the online Data Supplement Blood pressure, albuminuria Systolic blood pressure was measured with a computerized tail-cuff system (Process Control Blood Pressure 2900-series; TSE Systems, Bad Homburg, Germany) as described 2, 5. Mice were placed into metabolic cages for 6-hour urine collection. 25 Plasma and urine At the end of the experimental period heparinised blood was collected. Plasma urea-N (BUN) and cholesterol as well as urine creatinine were measured by an autoanalyzer (Hitachi 717; Roche, Mannheim, Germany). Albumin was measured by ELISA (Bethyl Laboratories, Montgomery, USA). Histopathologic analysis After removal kidney and heart tissue was fixed with 4% neutral buffered formalin, embedded in paraffin and sectioned at 1  m thickness. Sections were then deparaffinized and stained for light microscopy with PAS (Merck, Germany). Glomerular injury was evaluated histologically using a semi-quantitative scale with 0 indicating no injury, 1 mild injury in less than a third of the glomerular tuft, 2 damage of more than a third of the glomerular tuft, and 3 damage of the whole glomerulus. Under 200x magnification 20 glomeruli per animal were analysed 6. Renal infiltration by CD3 positive T cell, F4/80 positive monocyte/macrophages, GR-1 positive neutrophils and FoxP3 positive regulatory T cells was examined immunohistochemically as described 1. Cells were visualized immunohistochemically using antibodies specifically recognizing F4/80 (Clone BM8, product number T-2006, BMA Biomedicals, Augst, Switzerland), CD3 (polyclonal antibody, product number A 0452, DakoCytomation, USA), GR-1 (Ly6 G/C, NIMP-R14, Hycult Biotech, Netherlands) and FoxP3 (FJK-16s; eBiosciences, San Diego, CA). For detection ZytoChem-Plus AP Polymer kit was used (Zytomed, Berlin, Germany). Infiltration of CD3 and F4/80 cells was quantified by scoring 20 high power fields (0= 31 cells/field). Neutrophils and Tregs were quantified by counting the number of GR-1 or Foxp3 positive cells per field. Cardiac fibrosis was evaluated by scoring 6 1

3 Masson-trichrome stained heart sections using a scoring from 0 to Primary antibodies used for evaluation of autophagy were: guinea pig nephrin (1:100, Acris); rabbit Limp-2 (1:1000, Paul Saftig, Kiel, Germany); rabbit LC3B (1:100, Cell Signaling). All secondary antibodies used were fluorescent dye conjugated affinity purified donkey antibodies (Jackson ImmunoResearch). In brief, paraffin sections were deparaffinized and antigen retrieval was performed by microwave boiling (30 min, 800 W, 10 mM citrate buffer pH 6.1). Unspecific binding was blocked (5% horse serum, 0.05% triton X-100 in PBS, 30 min RT). 1°antibody incubations (in blocking buffer, o/n, 4°C) were followed by incubation with AF488 or Cy3 coupled 2°antibodies (1:400, 30 min, RT) and draq5 (Molecular Probes) for nuclear counterstain. Stainings were evaluated with an LSM 510 meta microscope using the LSM software (all Zeiss, Jena, Germany). Flow cytometry Previously described methods for leukocyte isolation from murine kidneys were used 1, 7. Kidneys were minced and digested with collagenase D and DNAse (Roche, Mannheim, Germany). Tubular fragments from digested kidneys were removed by percoll gradient centrifugation. Subsequently erythrocytes were lysed with ammonium chloride. For FACS analysis, fluorochrome-conjugated antibodies were used (CD45 (30-F11), CD3 (17A2), CD4 (GK1.5), CD8 (53-6.7)  -TCR (GL3), NK1.1 (PK136), CD11b (M1/70) and CD11c (HL3). Staining of intracellular IL-17A (TC11-18H10.1), IFNγ (XMG1.2) and IL-13 (eBio13A) was performed as recently described 1, 7. In brief, cells were activated by incubation for 4 h with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml; Sigma) and ionomycin (1 μg/ml; Calbiochem-Merck, Darmstadt, Germany). After 30 min of incubation, Brefeldin A (10 μg/ml; Sigma) was added. Samples were acquired on a Becton & Dickinson LSRII System using the Diva software. Data analyis was performed with FlowJo (Tree Star, USA) Real-time quantitative RT-PCR Total RNA from kidney cortex or heart ventricles was isolated using the RNeasy kit (Quiagen, USA). Real-time quantitative RT-PCR was performed using the Applied Biosystems ABI Prism system and SYBR Green JumpStart taq Ready Mix (Sigma, Germany). Mouse-specific PCR primers were used. The levels of mRNA expression in each sample were normalized to 18S expression 4, Statistics All data are expressed as mean  SEM. For the statistical analysis Graph Pad Prism 5.1 was used. 1-way ANOVA and post-hoc analyses by Newman-Keuls Multiple Comparison Test was performed.

4 References 1. Paust HJ, Turner JE, Steinmetz OM, Peters A, Heymann F, Holscher C, Wolf G, Kurts C, Mittrucker HW, Stahl RA, Panzer U. The IL-23/Th17 axis contributes to renal injury in experimental glomerulonephritis. J Am Soc Nephrol. 2009;20: Kirchhoff F, Krebs C, Abdulhag UN, Meyer-Schwesinger C, Maas R, Helmchen U, Hilgers KF, Wolf G, Stahl RA, Wenzel U. Rapid development of severe end-organ damage in C57BL/6 mice by combining doca salt and angiotensin II. Kidney Int. 2008;73: Sydow K, Schmitz C, von Leitner EC, von Leitner R, Klinke A, Atzler D, Krebs C, Wieboldt H, Ehmke H, Schwedhelm E, Meinertz T, Blankenberg S, Boger RH, Magnus T, Baldus S, Wenzel U. Dimethylarginine dimethylaminohydrolase1 is an organ-specific mediator of end organ damage in a murine model of hypertension. PLoS One. 2012;7:e Krebs C, Fraune C, Schmidt-Haupt R, Turner JE, Panzer U, Quang MN, Tannapfel A, Velden J, Stahl RA, Wenzel UO. CCR5 deficiency does not reduce hypertensive end-organ damage in mice. Am J Hypertens. 2012;25: Benndorf RA, Krebs C, Hirsch-Hoffmann B, Schwedhelm E, Cieslar G, Schmidt-Haupt R, Steinmetz OM, Meyer-Schwesinger C, Thaiss F, Haddad M, Fehr S, Heilmann A, Helmchen U, Hein L, Ehmke H, Stahl RA, Boger RH, Wenzel UO. Angiotensin II type 2 receptor deficiency aggravates renal injury and reduces survival in chronic kidney disease in mice. Kidney Int. 2009;75: Fraune C, Lange S, Krebs C, Holzel A, Baucke J, Divac N, Schwedhelm E, Streichert T, Velden J, Garrelds I, Danser AH, Frenay AR, van Goor H, Jankowski V, Stahl RA, Nguyen G, Wenzel UO. AT1 antagonism and renin inhibition in mice: Pivotal role of targeting angiotensin II in chronic kidney disease. Am J Physiol Renal Physiol. 2012;303: Riedel JH, Paust HJ, Turner JE, Tittel AP, Krebs C, Disteldorf E, Wegscheid C, Tiegs G, Velden J, Mittrucker HW, Garbi N, Stahl RA, Steinmetz OM, Kurts C, Panzer U. Immature renal dendritic cells recruit regulatory CXCR6(+) invariant natural killer T cells to attenuate crescentic GN. J Am Soc Nephrol. 2012;23:

5 Table S1 Basal data day 4 wildtype versus IL-17 knockout *=p<0.05, **=p<0.01, ***= p<0.001 vs. normotensive wildtype

6 Table S2 Basal data day 4 wildtype versus IL-17 knockout *=p<0.01 vs. normotensive wildtype

7 CD3 + cells (score) Neutrophils/ high power field F4/80 + cells (score) A B C ControlWildtype DOCA+Ang II IL-17 -/- DOCA+Ang II Figure S1 Early inflammation A and B Quantification of CD3 positive T cells and F4/80 positive macrophages showed increased infiltration in both hypertensive groups at day 4 but no significant difference between both genotypes. C The increase in GR-1 positive neutrophils was not significant at this timepoint. Figure S1 ControlWildtype DOCA+Ang II IL-17 -/- DOCA+Ang II ControlWildtype DOCA+Ang II IL-17 -/- DOCA+Ang II

8 Figure S2 Autophagy Representative confocal micrographs of double immunohistochemical stainings against the podocyte marker nephrin (A-F, red) and the autophagosomal marker LC3B (A-C, green) or the lysosomal marker Limp-2 (D-F, green) in controls and DOCA+Ang II treated wildtype or IL-17 -/- mice. An increased number of LC3 positive autophagosomes and Limp-2 positive lysosomes were found in podocytes of DOCA+Ang II mice with no difference between both genotypes.  = podocyte. LC3 / nephrin / draq5 10  m        Limp-2 / nephrin / draq5 10  m      Control Wildtype DOCA+Ang II IL-17 -/- DOCA+Ang II A B C D E F Figure S2

9 ANP (relative expression) Collagen I (relative expression) A B C Beta/alpha MHC (relative expression) Figure S3 Early cardiac gene expression A-C Real time RT-PCR analyses of mRNA revealed an upregulation of the fetal genes ANP and ratio of beta-MHC/alpha-MHC as well as collagen 1 but no significant difference was found between IL-17 -/- and wildtype mice. ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II Figure S3

10 Neutrophils/ high power field F4/80 + cells (score) A C Figure S4 Inflammation A and B An increased renal infiltration of F4/80 positive monocytes/macrophages and GR-1 positive neutrophils was found in hypertensive mice. Significantly more neutrophils but not monocyte/macrophages were found in IL-17 -/- compared to wildtype mice. C and D The IL-17 receptors were examined in a subset of the experiments. No regulation was found for IL-17a and c receptor expression in the kidney. E and F No significant difference was found between wildtype and IL-17 -/- mice for renal expression of VCAM-1 and IL-17F. G and H No significant difference was found between both genotypes for the number of infiltrating Foxp3 + cells and renal Foxp3 expression. *=p<0.01 ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II IL-17a receptor (relative expression) IL-17c receptor (relative expression) C ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II B ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II E VCAM-1 (relative expression) D ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II IL-17F (relative expression) F ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II Foxp3 (relative expression) Foxp3+ cells/ high power field ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II G H Figure S4

11 Figure S4 Renal lymphocytes in wildtype and IL-17 -/- mice I Quantification of renal lymphocytes. Cell surface staining of renal lymphocyte subsets stained for CD3, CD4, CD8 and NK1.1. J Representative cell surface staining for γδ TCR and CD3. K Intra- cellular staining for IFNγ and IL-13 of T cells isolated from the kidney revealed no increased TH1 or TH2 response in hypertension and no difference between wildtype and knockout mice. CD3 (% of all CD45 + cells) CD4 (% of all CD3 + cells) CD8 (% of all CD3 + cells) Double negative (% of all CD3 + cells) NK (% of all CD45 + cells) NKT (% of all CD3 + cells) Control4.1± ± ±2.78.4±0.95.9± ±0.0 Wildtype DOCA+Ang II 9.0± ± ±5.04.8±0.77.6± ±0.1 IL-17 -/- DOCA+Ang II 10.3± ± ±2.53.3±0.44.7± ±0.3 I IL-13 IFN-γ ControlWildtype DOCA+Ang II IL-17 -/- DOCA+Ang II K  TCR CD3 Control Wildtype DOCA+Ang II IL-17 -/- DOCA+Ang II J Figure S4 second page

12 A Figure S5 Characterization of renal macrophages and dendritic cells A Renal dendritic cells (CD11b +, CD11c + ) and macrophages (CD11b +, CD11c - ) were analyzed by flow cytometry in hypertensive wildtype and IL-17-deficient mice. B Qantification of dendritic cells and macrophages as defined in A. C Expression of F4/80, Ly6C and MHCII on renal dendritic cells and macrophages. CD11b CD11c ControlWildtype DOCA+AngII Dendritic cells F4/80 Cell count Ly6CMHCII Macrophages Dendritic cells Macrophages CD11b +, CD11c + (% of all CD45 + cells) CD11b +, CD11c - (% of all CD45 + cells) B C Wildtype DOCA+AngII IL-17 -/- DOCA+AngII Dendritic cells Macrophages B Control Wildtype DOCA+Ang II IL-17 -/- DOCA+Ang II Control Wildtype DOCA+Ang II IL-17 -/- DOCA+Ang II IL-17A -/- DOCA+AngII Control Figure S5

13 Relative heart weight (mg/g) ANP (relative expression) Cardiac fibrosis (score) Collagen I (relative expression) A B C D E Beta/alpha MHC (relative expression) Figure S6 Cardiac injury day 14 A An increased heart weight was found in the DOCA + Ang II mice. No difference was found between IL-17-deficient and wildtype mice. B No difference in cardiac fibrosis was seen between IL-17-deficient and wildtype mice as assessed by scoring. C-E Gene expression of ANP, MHC ratio and collagen type 1 was measured in 12 wildtype and 11 IL-17 -/- mice. All parameters were upregulated in hypertensive mice, but no significant difference between IL-17 -/- and wildtype mice was found. ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II ControlWildtype DOCA+Ang II IL17 -/- DOCA+Ang II Figure S6

14 IL-17F (relative expression) D Figure S7 Inflammation IL-23p19 -/- A Micrographs of renal Foxp3 immunohistochemistry slides are shown. B Counting of Foxp3 positive cells revealed a lower infiltration in IL-23p19 -/- than in wildtype mice but the difference was not significant. C In contrast, significantly lower renal expression of Foxp3 was found in hypertensive IL-23p19 deficient mice compared to wildtype mice. D Renal expression of IL-17F was not different in IL-23p19 -/- mice compared to wildtype mice. E Number of γδ T cells was significantly higher in IL-23p19 deficient than in wildtype mice. *=p<0.05 ControlWildtype DOCA+Ang II IL23p19 -/- DOCA+Ang II A ControlWildtype DOCA + Ang II IL-23p19 -/- DOCA + Ang II Foxp3+ cells/ high power field Foxp3 (relative expression) B C ControlWildtype DOCA+Ang II IL23p19 -/- DOCA+Ang II ControlWildtype DOCA+Ang II IL23p19 -/- DOCA+Ang II γδ T cells (% of all CD45 + cells) E ControlWildtype DOCA+Ang II IL23p19 -/- DOCA+Ang II Figure S7

15 ANP (relative expression) Collagen I (relative expression) A B C D Beta/alpha MHC (relative expression) Cardiac fibrosis (score) ControlWildtype DOCA+Ang II IL23p19 -/- DOCA+Ang II Figure S8 Cardiac injury IL-23p19 -/- A Scoring of Masson Goldner stained section revealed cardiac fibrosis without a significant difference between IL-23p19 -/- and wildtype mice. B-D Gene expression of ANP, the ratio of MHC isoforms and collagen type 1 is shown. All parameters were upregulated in hypertensive mice, but no significant difference between both genotypes was found. ControlWildtype DOCA+Ang II IL23p19 -/- DOCA+Ang II ControlWildtype DOCA+Ang II IL23p19 -/- DOCA+Ang II ControlWildtype DOCA+Ang II IL23p19 -/- DOCA+Ang II Figure S8

16 Glormerular in jury (score) Figure S9 Basal characterization IL-17 -/- and IL-23p19 -/- mice A PAS stained sections of wildtype, IL-17 -/- and IL-23p19 -/- mice are shown. No renal abnormalities were found. B This was confirmed by scoring. C No difference was found for infiltration of F4/80 + as well as CD3 + cells evaluated by immunohistochemistry. WildtypeIL-17 -/- IL-23p19 -/- Wildtype IL-17 -/- IL-23p19 -/- F4/80 + cells (score) CD3 + cells (score) Wildtype0.12± ±0.01 IL-17 -/- 0.07± ±0.04 IL-23p19 -/- 0.09± ±0.03 A B C Figure S9

17 Figure S10 Basal characterization of renal lymphocytes in wildtype and IL-17 -/- mice A Cell surface staining of renal lymphocyte subsets stained for CD3,  TCR, NK1.1, CD4 and CD8. B Quantification of the subsets. Wildtype  TCR NK1.1 CD4 CD3CD8CD3 IL-17 -/- CD3/  T cells NK/NKT cellsCD4/CD8 A CD3 (% of all CD45 + cells) CD4 (% of all CD3 + cells) CD8 (% of all CD3 + cells)  T (% of all CD45 + cells) Double negative (% of all CD3 + cells) NK (% of all CD45 + cells) NKT (% of all CD3 + cells) Wildtype12.8± ± ± ±0.16.6± ± ±0.3 IL-17 -/- 12.9± ± ± ±0.16.0± ± ±0.2 B Figure S10

18 A Figure S11 Characterization of renal macrophages and dendritic cells A Renal dendritic cells (CD11b +,CD11c + ) and macrophages (CD11b +,CD11c - ) were analyzed by flow cytometry in healthy wildtype and IL-17-deficient mice. B Qantification of dendritic cells and macrophages as defined in A. C Expression of F4/80, Ly6C and MHCII on renal dendritic cells and macrophages. CD11b CD11c Wildtype IL-17 -/- Dendritic cells F4/80 Cell count Ly6CMHCII Macrophages Dendritic cells Macrophages CD11b + CD11c + (% of all CD45 + cells) CD11b + CD11c - (%of all CD45 + cells) B C Wildtype IL-17 -/- Dendritic cells Macrophages B WildtypeIL-17 -/- WildtypeIL-17 -/- Figure S11


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