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T-regulatory Cells in Renal Ischemic injury Alvaro Pacheco-Silva Laboratory of Clinical and Experimental Immunology Division of Nephrology Universidade Federal de São Paulo Hospital do Rim e Hipertensão Hospital Israelita Albert Einstein São Paulo, Brasil Ischemia and Reperfusion Injury Very complex Incompletely understood Ischemic phase - blockade of blood influx, oxygen and nutrients Reperfusion phase – enhancement of tissue damage Interaction between vasculature (endothelium), tubular cells and incoming cells. Acute Renal Failure (ARF) Delayed Graft Funciton (DGF) Graft Rejection Bonventre & Weinberg, JASN 14:2199-2210, 2003; Schrier et al, J Clin Invest 14(1):5-14, 2004 Ischemia/Reperfusion Injury Oxygen deprivation Reactive oxygen species (ROS) Cellular events Cytoskeletal breakdown Loss of polarity Apoptosis and necrosis Desquamation of viable and necrotic cells Tubular obstruction Inflammatory response Endothelial activation Leukocyte activation and migration – neutrophils, lymphocytes, macrophages Cytokines, chemokines Adhesion molecules Haemodynamic events Vasoconstriction Vasodilatation Endothelial and smooth muscle cells structural damage Endothelial-leukocyte adhesion, vascular obstruction Immune response in IRI Boros and Bromberg, Am J. Transplantation 2005 Impact of ischemia/Reperfusion injury Acute Rejection 45 Rejection (%) 40 35 30 25 Renal Fibrosis P 0,01 NO DGF With DGF P 0,01 20 15 10 5 0 Discharge 6 months > 6 months Ojo et al. Transplantation 1997. Burne-Tarne al. Kidney Int 2005. Prediction of Clinical outcomes (DGF) Variables p value Donor type <0,001 CIT 0,005 WIT 0,013 TNFα <0,001 CD25 <0,001 TGF-β <0,001 A20 <0,001 IL-10 <0,001 ICAM 0,006 CD4+T lymphocytes and IRI A phase I trial of immunossupression with anti-ICAM-1 (CD54) mAb in renal allograft recipients. Haug C. et al. Transplantation 55(4):766-772, 1993. Graft Treatment Primary Non Function Graft Survival Kidney BIRR1 (anti-ICAM) 0 78 % Contralateral Kidney Current immunosuppression 3 56 % N= 18 hight risk for delayed graft function patients Follow up 16 to 30 months CD4+T lymphocytes and IRI A prospective, randomized, clinical trial of intraoperative versus postoperative Thymoglobulin in adult cadaveric renal transplant recipients. Goggins WC. et al. Transplantation. 76(5):798-802, 2003 Fig. 1 Fig. 2 Post Op Days CD4+T lymphocytes participates on IR injury Identification of the CD4+T cell as a major pathogenic factor in ischemia acute renal failure. Burne MJ. Et al. J Clin Invest. 2001 Nov;108(9):1283-90. b a WT type WT type Nu/nu mice CD4 -/- Nu/nu mice reconstituted wild-type T cells CD4-/reconstituted with wild-type T cells Strategies of treatment IRI INJURY PROTECTIVE MECHANISMS Tregs and Innate immune response Enhanced Regulatory T Cell Activity is an Element od the Host response to Injury Choileain NN. Et al. J immunol 2006 Jan 1;176(1):225-36 Regulatory T cells Developmental classification for T Reg Naturally arising T reg cells: Adaptative T regs: Constitutive expression at higher levels: Interleukin (IL)-2 receptor alpha chain (CD25) Induced from naive T cells by antigenic stimulation and cytokine millieu (Sakaguchi et al., 2004) CTLA-4 (CD152) (Sakaguchi et al., 2004) Glucocorticoid induced TNF receptor (GITR) (McHugh et al., 2002, Shimizu et al., 2002) FOXP3 (Schubert and Ziegler et al, 2001, Fontenot et al, 2003; Hori et al, 2003) IL-10 (Roncarolo et al, 2006) TGF-beta (Nakamura et al, 2004) CD4+T lymphocytes in Renal Ischemia reperfusion model Strategies CD4+ T cells CD4+CD25+Foxp3+ T Cells Decrease number (Anti-CD25 depleting antibody) Decrease suppressor activity (Anti-GITR) Post ischemic injury CD4+ effector T Cells Increase CD4+ effector T cells activity Post ischemic injury ? PC61: depleting rat IgG1 anti-CD25 (alpha chain of IL-2R) Choileain NN. Et al J. Immunol, 2006 DTA-1: agonist rat IgG2a anti-GITR (Glucocorticoid-induced TNF receptor) DTA-1 mAb abrogates suppression mediated by CD25+CD4+ T cells breaking immunological self- tolerance DTA-1 mAb abrogates suppression mediated by CD25+CD4+ T cells leading to development of autoimmune gastritis in mice IR antibody treatment Protocol Sacrifice: Blood, kidneys, spleen, lymph nodes harvested Treatment PC61 200 g DTA-1 400 g IgG 400 g Day 0 Isquemia 45 min Day 1 Day 2 (24 hs) Day 4 (72hs) Renal Ischemia Reperfusion Model Background: C57 BL/6 male cm Microclamps for renal artery and vein (renal pedicle) IRI model adapted from KELLY et al., 1996 Reperfusion times : 24 and 72hs Analyses: blood: creatinine and urea LN, spleen: flow cytometry Kidneys: morphometry, Real Time PCR IR antibody treatment Effect of Antibody treatment in TCD4+CTLA4+Foxp3+ cells PC61 Treatment DTA-1 Treatment Depletion of TCD4+CTLA4+FOXP3+ cells by PC61 treatment 100 IR72 32,2 20 % depletion % depletion IR24 40,2 50,1 50 69,6 60 0 60 90,2 80 40 Effect of by by DTA-1 treatment in the TCD4+CTLA4+Foxp3+ population 40 30 20 IR24 14,0 7,6 10 SPl LN Organ 0 13,2 SPl LN Organ IR72 IR antibody treatment Depletion of TCD4+CTLA4+Foxp3+ cells IgG PC61 IR 24 12.62 PC61 IR 72 10.52 8.08 3.11 1.57 Spleen 18.22 pRenal LN IR antibody treatment Renal Function Outcome weight loss after IR24 + antibody treatment Blood Urea Weight Loss (g) P=0,0002 200 160 140 120 100 80 P=0,0 018 IgG IgG PC 61 DTA-1 PC61 DTA-1 Treatment 60 weight loss after IR 72 + antibody treatment 40 20 P=0,0033 0 24 72 Hours 7 Weight Loss (g) Blood Urea (mg/dL) 180 4 3,5 3 2,5 2 1,5 1 0,5 0 6 P=0,000 1 5 4 3 2 1 0 IgG PC61 Treatment DTA-1 Morphometric analyses Acute tubular necrosis and tubular regeneration IR 24 hours Morphmetric analy ses IR 24 groups * 80 * 70 Score 60 * 50 necrosis 40 regeneration 30 20 10 0 IgG IR 24 PC61 IR 24 Groups * p< 0.001 DTA-1 IR 24 Morphometric analyses Acute tubular necrosis and tubular regeneration IR 72 hours Morhmetric analyses IR72 groups * 70 60 * Score 50 * 40 necrosis 30 regeneration 20 10 0 IgG IR 72 PC61 IR 72 Groups * p< 0.001 DTA-1 IR 72 IR antibody treatment Histopathological analyses HE Sham IgG PC61 200 g DTA-1 400 g D A B C E F G 24 H I H 72 H Kidney Tissue HE. A, B, C, D: IR 24 hs. A: Sham, B: IgG treated, 400 micrograms C: PC61 treated, 200 micrograms. D: DTA-1 treated, 400 micrograms, E, F, G, H: IR 72 hs. E: Sham, F: IgG treated, 400 micrograms, G: PC61 treated 200 micrograms mg, H: DTA-1 treated, 400 microgams. IR antibody treatment Anti-Inflammatory Genes/TH2 response GATA-3 Relative Expression PC61 HO-1 Relative Expression PC61 6 Gata-3 HO-1 5 4 3 2 1 0 NR 4 3,5 3 2,5 2 1,5 1 0,5 0 NR IgG IR PC61 IR IgG IR PC61 IR 24 24 72 72 HO-1 Gata-3 IgG IR DTA-1 IgG IR DTA-1 24 IR24 72 IR72 Groups IgG IR 72 PC61 IR 72 GATA-3 Relative Expression DTA-1 HO-1 Relative Expression DTA-1 NR PC61 IR 24 Groups Groups 40 35 30 25 20 15 10 5 0 IgG IR 24 8 7 6 5 4 3 2 1 0 NR IgG IR 24 DTA-1 IR24 Groups IgG IR 72 DTA-1 IR72 IR antibody treatment Pro-Inflammatory Genes IL-1b Relative Expression PC61 IL6 Relative Expression PC61 5 IL-1b IL6 4 3 2 1 5 4 3 2 1 0 NR 0 NR IgG IR 24 PC61 IR 24 IgG IR 72 PC61 IR 72 IgG PC61 IgG PC61 IR 24 IR 24 IR 72 IR 72 Groups Groups IL-1b Relative Expression DTA-1 1,4 1,2 1 0,8 0,6 0,4 0,2 0 20 IL-1b IL6 IL6 Relative Expression DTA-1 15 10 5 0 NR NR IgG IR 24 DTA-1 IR24 Groups IgG IR 72 DTA-1 IR72 IgG IR DTA-1 IgG IR DTA-1 24 IR24 72 IR72 Groups IR antibody treatment Foxp3 Relative Expression PC61 TGFb Relative Expression PC61 5 TGFb Foxp3 4 3 2 1 75 60 45 30 15 0 NR 0 NR IgG PC61 IgG PC61 IR 24 IR 24 IR 72 IR 72 Foxp3 Relative Expression DTA-1 TGFb Foxp3 4 2 NR IgG DTAIgG DTAIR 24 1 IR 72 1 IR24 IR72 Groups PC61 IR 72 75 60 45 30 15 0 NR 0 IgG IR 72 TGFb Relative Expression DTA-1 10 6 PC61 IR 24 Groups Groups 8 IgG IR 24 IgG DTA- IgG DTAIR 24 1 IR 72 1 IR24 IR72 Groups IR antibody treatment Conclusions and Perspectives At 24 hours of reperfusion, depletion of TCD4+CTLA-4+Foxp3+ cells was 30,3% (spleen) and 67,8% (para renal lymphnodes).After 72 hours of reperfusion, depletion of TCD4+CTLA-4+Foxp3+ was 43,1% (spleen) and 90,22% (para renal lymphnodes). This depletion was efficient in generate significant responses in both 24 hours and 72 hours if reperfusion Depleted mice presented similar renal function to control animals at 24 hours, but 72 hours after IRI, PC61 treated mice presented significant worst renal function compared to the group that received IgG. DTA-1 treated animals presented significant protection at the same timepoint, indicating that different subsets of cells can be acting at these timepoints. Furthermore, histopathological analyses showed that there was a pronounced incidence of necrosis for both PC61 treated and IgG in IR 24 hours experiments. On the other hand, in IR 72 hours experiments we observed a regeneration pattern in both PC61 and IgG treated animals, but in the PC61 treated group there was a significant necrosis index (p<0.001), comparing with IgG treated group, suggesting that TCD4+CTLA4+FOXP3+ cell population could be important in a late phase of injury recover. It is known that the stress and tissue damage associated with IRI influence the development of a immune response to protect the tissue damage. Thus, our results suggests a role for TCD4+CTLA4+FOXP3+ cells (naturally arising T teg cells) in renal IRI experimental model. IR antibody treatment Hypothesis CD4+CD25+Foxp3+ T Cells CD4+ effector T Cells 24 hs: TCD4+ effector Stimulated by DTA-1 escape from T Reg suppression and make injury worst 72 hs: TRegs GITRhigh stimulated by DTA-1 start to suppress TCD4+ response 72 hs: If there is no T Reg at this point (PC61 treatment), there is no recovery from injury Post ischemic injury Proinflamatory cytokines IL-1b and IL-6 Anti inflamatory genes Such as HO-1 and polarization toward Th2 (GATA-3) transcription factor Persistence of necrosis IR antibody treatment Perspectives Acknowledgements Rebecca M. M. Monteiro Marcio J. Damião Giselle Gonçalves Carla Q. Feitoza Marcos Cenedeze Nephrology Division Universidade Federal de São Paulo Brazil Prof. Dr.Mauricio M. Rodrigues Fanny Tzelepis Interdisciplinary Center for Gene Therapy CINTERGEN Universidade Federal de São Paulo, Brazil Prof. Dr.Niels Olsen S. Camara Immunology Division Universidade de São Paulo USP, Brazil Vicente de Paula A. Teixeira Marlene A. dos Reis Department of Pathology, Universidade Federal de Uberaba, Minas Gerais, Brazil Prof Dr. S. Sakaguchi T. Yamaguchi (DTA-1) H. Uryu (PC61) K. Nagahama (IRI) M. Ono (Real Time PCR) Institute for Frontier Medical Sciences Kyoto University Japan ã