Universidade Federal do Rio de JaneiroCentro de Ciências da Saúde

Instituto de Biofísica Carlos Chagas Filho

The importance of apoptosis for immune regulation in Chagas’ disease

George A. DosReis

Fabrício Montalvão. IBCCF-UFRJLandi V. Guillermo. IBCCF-UFRJ

Geisy M. de Almeida. IBCCF-UFRJElizabeth M. Silva. IBCCF-UFRJ

Marise P. Nunes. IOC-Fiocruz-RJJuliana de Meis. IOC-Fiocruz-RJ

Christina M. Takiya. Dept. Histologia-UFRJRichard Siegel. NIAID-NIH, USA

Celio Freire-de-Lima. IBCCF-UFRJMarcela F. Lopes. IBCCF-UFRJ

Lopes MF et al. Immunol. Today 21; 489-494, 2000Freire-de-Lima CG et al. Nature 403; 199-203, 2000

Phagocytosis of apoptotic cells (efferocytosis) inactivates macrophages and exacerbates parasite replication through TGF-β, ODC and polyamine synthesis

X

Efferocytosis

T. cruzi infection: Consequences of apoptosis

Efferocytosis

Apoptosis in T. cruzi infection

Modify efferocytosis by:

1. Caspase inhibition2. Anti-FasL treatment3. Antibodies to apoptotic cells

Efferocytosis

Blocking apoptosis in T. cruzi infection

Caspase inhibition

Efferocytosis

Caspase inhibition reduces lymphocyte apopotosis in vivo

Silva, E. M. et al. Caspase inhibition reduces lymphocyte apoptosis and improves host immune responses to Trypanosoma cruzi infection. Eur. J. Immunol. 2007, 37 (3), 738-746.

Treatment with zVAD reduces parasitemia

10 15 20 25 30 35 400

10

20

30

40

50

60

70

80

90

100

zVAD

zFA

control

Days post infection

Par

asit

es x

10

4/m

l

10 15 20 25 30 35 400

10

20

30

40

z-VAD

z-FAcontrol

Days post infectionP

aras

ites

x 1

04/m

l

Silva, E. M. et al. Caspase inhibition reduces lymphocyte apoptosis and improves host immune responses to Trypanosoma cruzi infection. Eur. J. Immunol. 2007, 37 (3), 738-746.

Treatment with zVAD in vivo increases macrophage activation

The general caspase inhibitor zVAD

reduces apoptosis of T lymphocytes in vitro

and in vivo

Treatment with zVAD increases immunity

to T. cruzi infection

Blocking apoptosis in T. cruzi infection

Anti-FasL

Efferocytosis

Increased immunity to T. cruzi after treatment with anti-FasL in vivo

Guillermo, L.V. et al. The Fas death pathway controls coordinated expansions of type 1 CD8 and type 2 CD4 T cells in Trypanosoma cruzi infection. J. Leukoc. Biol. 2007, 81, 942-951.

Anti-FasL in vivo accelerates and increases type 1 cytokine responses

Anti-FasL reduces apoptosis of CD4 and CD8 T cells

Anti-FasL increases type 1 cytokine responses, increases macrophage NO production and reducesparasitemia

Lymphocyte apoptosis is deleterious for T. cruziInfection in vivo

Modifying immune regulation throughopsonization of apoptotic cells

Antibodies

Efferocytosis

Differential reaction of chagasic IgG with apoptotic lymphocytes

1 10 100 1000

0

20

40

60

80

100

% o

f M

ax

Annexin V+

CHACT - 26.1%

CHA - 36.3%

IgG (H+L)

A

10000

OD (Arbitrary Units)

Migration Distance (Arbitrary Units)

CT

400 600 800 1000

50

40

30

20

10

0

B

C

Position 403

Pea

k va

lues

(A

U)

Position 901 Position 1055Position 1022

Cha

0

2

4

6

8

10Parasite number (x104)

CT CHA CT CHA

None IgG IgM

None APO

**

Opsonization of apoptotic cells with chagasic IgG reduces the effect of efferocytosison replication of T. cruzi

IgG purified from T. cruzi infection(90 days) or age matched serum

Control of parasite replication is mediated by increased TNF-α production

Apoptotic cells opsonized with chagasic IgGincrease TNF-α production

02468

101214

NoneAnti-TNF-Rat IgG1

Parasite number (x10 5)

CT CHA None IgG

None APO

** **

0

200

400

600

800

1000

1200

NoneAPOAPO/CT IgGAPO/CHA IgG

TNF- (pg/ml)

**

A

B

Control of parasite replicationrequires engagement of FcγRs

0

3

6

9

12

15

ControlChagasic

Parasite number (x104)

IgG IgG F(ab') 2

None APO

**

0

4

8

12

16

20Control FabCD16/CD32 Fab

Parasite number (x104)

Control Chagasic

None IgG APO

**

A

B

Previous immunization with apoptotic cells reduces parasitemia in vivo

0 5 10 15 200

1020304050607080

Saline/Al(OH)3APO/Al(OH)3

Parasitemia (x104/mL)

**

Dm28c

dpi

Conclusions Chagasic IgG opsonizes apoptotic cells and reduces the effect of efferocytosis on parasite replication

The protective effect is mediated by increased TNF-α production

The protective effect requires FcγRs

Generation of autoantibodies against apoptotic cell antigens could play a protective role against parasite infection