Transcript T-regulatory cells in ischemic injury.

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
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