Inhibition of caspase-8 activity reduces IFN-γ expression by T cells from Leishmania major infection

Following infection with Leishmania major, T cell activation and apoptosis can be detected in draining lymph nodes of C57BL/6-infected mice. We investigated the mechanisms involved in apoptosis and cytokine expression following T cell activation. After twoweeks of infection, apoptotic T cells were not detected in draining lymph nodes but activation with anti-CD3 induced apoptosis in both CD4 and CD8 T cells. Treatment with anti-Fas Ligand, caspase-8 or caspase9 inhibitors did not block activation-induced T-cell death. We also investigated whether the blockade of caspase-8 activity would affect the expression of type-1 or type-2 cytokines. At early stages of infection, both CD4 and CD8 T cells expressed IFN-γ upon activation. Treatment with the caspase-8 inhibitor zIETD-fmk (benzyl-oxycarbonyl-IleGlu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone) reduced the proportion of CD8 T cells and IFN-γ expression in both CD4 and CD8 T cells. We conclude that a non apoptotic role of caspase-8 activity may be required for T cell-mediated type-1 responses during L. major infection.


INTRODUCTION
Immunity to Leishmania infection correlates with protective type 1 response in resistant hosts.CD4 Th1 cells have been considered as a major source of IFN-γ , with NK cells and CD8 T cells playing a minor role in primary infection (McMahon-Pratt and Alexander 2004).Susceptibility to Leishmania major infection has been associated with type 2 cytokine responses; whereas IL-10 contributes to parasite persistence (Belkaid et al. 2002a), the role of IL-4 produced by CD4 Th2 cells remains controversial (Mohrs et al. 1999, Noben-Trauth et al. 1999, Noben-Trauth et al. 2003).
Apoptosis of both CD4 and CD8 T cells has been observed within cutaneous lesions in human leishmaniasis (Bertho et al. 2000), as well as in lymph nodes from mice infected with L. major (Desbarats et al. 2000).Apoptosis contributes to T cell anergy to Leishmania amazonensis antigens (Pinheiro et al. 2004), but the mechanisms involved in T cell death in cutaneous leishmaniasis are not known.
T cell apoptosis can be mediated by intrinsic pathway involving growth factor deprivation and caspase-9 activation (Alves et al. 2007).By contrast, activationinduced cell death (AICD) occurs upon induction of Fas Ligand (FasL) expression, interaction with death receptor Fas and recruitment of caspase-8 (Lenardo et al. 1999, Krammer 2000).Caspase-8 is also involved in T cell signaling for proliferation, IL-2 production and development of CD8 T cell memory (Alam et al. 1999, Kennedy et al. 1999, Chun et al. 2002, Wu et al. 2004).
We investigated the role of caspase-8 in T-cell apoptosis and control of cytokine responses in draining lymph nodes upon L. major infection.Activation-in-An Acad Bras Cienc (2008)  duced T cell death occurs in both CD4 and CD8 T cells from infected mice.Nonetheless, inhibitors of FasL, caspase-8 or caspase-9 did not affect T cell apoptosis, whereas caspase-8 inhibition reduced IFN-γ expression upon stimulation of T cells.These results suggest that caspase-8 activity may be required in the development of protective immune responses to Leishmania infection.

MICE AND L. major INFECTION
Female C57BL/6 mice were obtained from the Federal University of Rio de Janeiro.L. major LV39 isolated from BALB/c mice were cultured in Schneider's medium at 28 • C during 4 days and used at the stationary phase of the culture.Mice, aging 7-9 wk, were infected in the left hind footpad with sc injection of 3 × 10 6 metacyclic promastigotes/30 µL.Mice were killed during the acute phase at 2, 6, and 13 weeks post infection.All experiments and animal handling were conducted according to approved institutional protocols.

FLOW CYTOMETRY
Fresh or cultured cells were washed in sorting buffer (containing 2% FBS) and incubated with anti-CD16/ CD32 for Fc blocking, followed by staining with allophycocyanin-labeled anti-CD8 or anti-CD4 for 30 min at 4 • C. All mAbs used in flow cytometry are from BD Pharmingen.For apoptosis detection, cells were washed to remove excess of surface staining reagents, and then stained with FITC-annexin V (apoptosis detection kit; R&D Systems) for 20 min at room temperature in annexin buffer or with 7-AAD, which was added just prior to flow cytometry.Cells were also stained with FITClabeled anti-CD44 as a marker for T cell activation.Cells were washed and acquired on a FACSCalibur system, by using Cell quest software (BD Biosciences).For analysis, FlowJo software was used (TreeStar).

DATA ANALYSIS
Results are expressed as average and SEM in figures and the number (n) of animals per group was indicated in figure legend.For in vitro experiments, data are expressed as average of 2-3 determinations per treatment, in each of at least 3 repeat experiments, and significant differences detected in Student's t test were shown for p < 0.05( * ).

T CELL APOPTOSIS DURING L.major INFECTION
We infected B6 mice in the footpads with L. major and followed the onset of activation and apoptosis in T cells from draining (popliteal) lymph nodes.Increased ex- pression of CD44 and staining with 7-AAD were assessed as markers of activation and apoptosis in T cells, respectively.We observed increased activation and apoptosis 6 wk upon L. major infection in both CD4 and CD8 T cells (Fig. 1).After 13 wk of infection, however, activation returned to baseline levels (Fig. 1A and  B), whereas apoptosis remained elevated (Fig. 1C and  D).We investigated the mechanisms of T cell apoptosis in vitro.Activation with anti-CD3 induced apoptosis in both CD4 and CD8 T cells from draining lymph nodes as early as 2 wk (Fig. 2A and B) upon infection with L. major.AICD was more evident in CD4 than in CD8 T cells upon 2 (Fig. 2A and B) or 6 (Fig. 2C and D) wk of infection.To investigate the role of Fas-death path-way, we treated T cell cultures with anti-FasL or control IgG antibody and assessed apoptosis by annexin V staining upon 24 h.Treatment with anti-FasL did not inhibit apoptosis induced upon T cell activation (Fig. 3A and  B).To address whether caspase-8 or caspase-9 were involved in apoptosis, T cells were activated in the presence of caspase-8 (zIETD) or caspase-9 (zLEHD) inhibitor.Neither zIETD nor zLEHD inhibited AICD in T cells from L. major infection (Fig. 3C and D).

CASPASE-8 AND CYTOKINE EXPRESSION BY T CELLS
Previous experiments suggest a non apoptotic role of caspases in T cell signaling for cytokine responses (Sehra et al. 2005, Su et al. 2005).We addressed by flow cyto- metry cytokine expression in T cell subsets, and whether caspase-8 controls type-1/type-2 cytokines upon L. major infection.First, T cells from draining lymph nodes (2 wk post infection) were activated with anti-CD3 in the presence of caspase-8 inhibitor zIETD or DMSO only during 72 h.Then, T cells were re-stimulated with ionomycin and PMA, in the presence of brefeldin A, to prevent cytokine secretion.The presence of zIETD did not affect the number of cells recovered from T cell cultures (not shown).The proportions of CD4 and CD8 T cells were similar (30-40%) in cultures (Fig. 4A and B).Caspase-8 inhibition negatively affected CD8, but not CD4, T cells (Fig. 4A and B) and the proportion of cells expressing IFN-γ , but not IL-10 (Fig. 4C and D).Both CD4 and CD8 T cells expressed IFN-γ (Fig. 5A and B), whereas expression of IL-4 was not detected even upon culture with exogenous IL-4 (not shown).The inhibition of caspase-8 decreased the expression of IFN-γ in both CD4 and CD8 T cells (Fig. 5A and B).These results suggest that caspase-8 plays a role in the control of cytokine responses to L. major infection.

DISCUSSION
Apoptosis of CD4 T cells has been suggested as a mechanism to reduce Th1 responses in Leishmania donovani infection (Das et al. 1999).In addition, Fas and/or FasL expression has been detected in T cells from lymph nodes (Desbarats et al. 2000) or lesions (Eidsmo et al. 2005) during L. major infection, and mice with defective Fas/ FasL expression show increased Th1 responses upon L. donovani (Alexander et al. 2001) andL. major (Huang An Acad Bras Cienc (2008)  80 (1) et al. 1998) infections.We observed increased apoptosis in both CD4 and CD8 T cells from draining lymph nodes only at later stages of L. major infection.T cell apoptosis can also be induced by stimulation in vitro (AICD) as early as 2 wk and after 6 wk, at the peak of lymphocyte activation in vivo.Nonetheless, AICD was not blocked by inhibitors of FasL, caspase-8 or caspase-9, although these reagents have been successfully used at the same doses in previous unrelated experiments (Silva et al. 2005, Guillermo et al. 2007).Therefore, AICD in lymph nodes from L. major infection may be mediated by other mechanisms, such as granzyme-B-induced apoptosis (Devadas et al. 2006).
By contrast, caspase-8 inhibition by zIETD peptide negatively affected CD8 T cells and the expression of IFN-γ in T cells from L. major-infected mice.Similarly, CD8 T cells have defective survival and IFN-γ expression in mice bearing a T cell-restricted transgene for the viral-FLICE/caspase-8 inhibitory protein (vFLIP) (Wu et al. 2004).These results suggest a paradoxical role of caspase-8 preventing, rather than inducing T cell death.Although caspase-8 inhibition could affect CD8 T-cell responses during L. major infection, the role of CD8 T cells as an important source of IFN-γ in the immunity to primary L. major infection is disputed (Huber et al. 1998, Belkaid et al. 2002b).However, it has been suggested that CD8 T cells can favor IFN-γ expression by protective CD4 Th1 cells during L. major infection.(Herath et al. 2003).
Based on evidence suggesting a non apoptotic role of caspase-8 in T cell signaling for cytokine expression (Su et al. 2005), we studied how caspase-8 inhibition would affect the development of type-1 vs type-2 responses following L. major infection.Caspase-8 inhibition decreased the expression of IFN-γ by both CD4 and CD8 T cells.It is possible that these effects are due to defective signaling for cytokine expression upon caspase-8 inhibition as previously observed for NF-κB activation and IL-2 secretion (Silva et al. 2005, Su et al. 2005).In agreement with this idea, defective NF-κB activation affects negatively both proliferation and the production of IFN-γ by T cells (Corn et al. 2003).By contrast, IL-10 expression in T cells from L. major infection was not affected by inhibition of caspase-8 even in the presence of IL-4 (not shown).Therefore, non apoptotic activity of caspase-8 is not necessary for the expression of type-2 cytokines, but may be required for the induction of protective type-1 response in L. major infection.
Fig. 1 -T cell activation and apoptosis during L. major infection.Lymph node cells from normal and L. major-infected mice were analysed by flow cytometry.Activation of CD4 (A) and CD8 (B) T cells was determined by high expression of CD44.Apoptosis was assed upon staining with 7-AAD in CD4 (C) and CD8 (D) T cells.Open bars represents T-cell activation and apoptosis in a pool of (inguinal and popliteal) lymph nodes from normal mice, black bars represent draining (popliteal) lymph nodes from L. major-infected mice.Results are expressed as average and SEM (n=2-3 mice/group).

Fig. 2 -
Fig. 2 -Activation-induced cell death upon infection with L. major.Lymph node cells from normal and L. major-infected mice were cultured in the presence of medium only (open bars) or stimulated with anti-CD3 (black bars).After 24 h, cells were stained with anti-CD4 (A and C), anti-CD8 (B and D), 7-AAD and evaluated by flow cytometry.Panels show percentages of apoptotic (7-AAD + ) CD4 and CD8 T cells upon 2 (A and B) or 6 (C and D) weeks of infection.Results are expressed as average and SEM.Significant differences between cells treated with medium and anti-CD3 were indicated for p < 0.05( * ).

Fig. 3 -
Fig. 3 -T-cell death pathways during L. major infection.Lymph node cells from infected (2 wk) mice were cultured with medium only (open bars) or stimulated with anti-CD3 (black bars) in the presence of anti-FasL or control IgG (A and B) or in the presence of zIETD, zLEHD or stock diluent DMSO (C and D).After 24 h, cultured cells were stained with anti-CD4, anti-CD8, and annexin V. Panels show percentages of apoptotic (annexin V + ) CD4 (A and C) and CD8 (B and D) T cells.Results are expressed as average and SEM.

Fig. 4 -
Fig.4-Expression of type 1 and type 2 cytokines in L. major infection.Lymph node cells from infected (2 wk) mice were stimulated with anti-CD3 only or in the presence of zIETD or stock diluent DMSO for 72 h.Cells were re-stimulated with PMA and ionomycin for 4 h, in the presence of brefeldin A. Cells were stained with anti-CD4 (A), anti-CD8 (B) or permeabilized and stained with anti-IL-10 (C) and anti-IFN-γ (D).Results are expressed as average and SEM.Significant differences between cells treated with zIETD and DMSO were indicated for p < 0.05( * ).

RESUMOAFig. 5 -
Fig. 5 -Inhibition of caspase-8 reduces IFN-γ expression by T cells from L. major infected mice.Lymph node cells from infected (2 wk) mice were stimulated with anti-CD3 in the presence of zIETD (right panels) or stock diluent DMSO (left panels), re-stimulated as described above and stained with anti-CD4 (A), anti-CD8 (B) and anti-IFN-γ (A and B).Panels show percentages of IFN-γ + CD4 T cells (A), and IFN-γ + CD8 T cells (B) as assessed by flow cytometry.