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

Abstracts

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 Tcellactivation. After two weeks 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-FasLigand, caspase-8 or caspase- 9 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-gamma upon activation. Treatment with the caspase-8 inhibitor zIETD-fmk (benzyl-oxycarbonyl-Ile- Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone) reduced the proportion of CD8 T cells and IFN-gamma expression in both CD4 and CD8T 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.

apoptosis; caspase-8; CD8 T cells; IFN-gamma; Leishmaniasis

A ativação e a morte por apoptose de linfócitos T foram observadas em linfonodos drenantes de camundongos C57BL/6 infectados com Leishmania major. Investigamos os mecanismos envolvidos na apoptose e na expressão de citocinas após a ativação de linfócitos T. Após duas semanas de infecção, embora as células apoptóticas ainda não sejam detectadas em linfonodos drenantes, células T CD4 e CD8 sofrem apoptose após ativação com anti-CD3. O tratamento com anticorpo antagonista anti-Ligante de Fas, ou com inibidores das caspases-8 e 9, não bloqueou a morte induzida por ativação das células T. Investigamos também se a inibição da atividade da caspase-8 poderia afetar a expressão de citocinas tipo-1 ou tipo-2. Nos estágios iniciais da infecção, células T CD4 e CD8 de animais infectados com L. major expressaram IFN-gama após ativação. O tratamento com o inibidor de caspase-8 zIETD (benzoil-oxicarbonil-Ile-Glu(OMe)-Thr-Asp(OMe)-fluorometilcetona) durante a estimulação de células T reduziu a proporção de células T CD8 e a expressão de IFN-gama por células T CD4 e CD8. Concluimos que a atividade não apoptótica de caspase-8 pode ser necessária para o estabelecimento da imunidade mediada por células T durante a infecção por L. major.

apoptose; caspase-8; células T CD8; IFN-gama; Leishmanioses

BIOMEDICAL AND MEDICAL SCIENCES

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

Wânia F. Pereira; Landi V.C. Guillermo; Flávia L. Ribeiro-Gomes; Marcela F. Lopes

Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, Ilha do Fundão, 21941-902 Rio de Janeiro, RJ, Brasil

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ABSTRACT

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 Tcellactivation. After two weeks 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-FasLigand, caspase-8 or caspase- 9 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-g upon activation. Treatment with the caspase-8 inhibitor zIETD-fmk (benzyl-oxycarbonyl-Ile- Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone) reduced the proportion of CD8 T cells and IFN-g expression in both CD4 and CD8T 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.

Key words: apoptosis, caspase-8, CD8 T cells, IFN-g , Leishmaniasis.

RESUMO

A ativação e a morte por apoptose de linfócitos T foram observadas em linfonodos drenantes de camundongos C57BL/6 infectados com Leishmania major. Investigamos os mecanismos envolvidos na apoptose e na expressão de citocinas após a ativação de linfócitos T. Após duas semanas de infecção, embora as células apoptóticas ainda não sejam detectadas em linfonodos drenantes, células T CD4 e CD8 sofrem apoptose após ativação com anti-CD3. O tratamento com anticorpo antagonista anti-Ligante de Fas, ou com inibidores das caspases-8 e 9, não bloqueou a morte induzida por ativação das células T. Investigamos também se a inibição da atividade da caspase-8 poderia afetar a expressão de citocinas tipo-1 ou tipo-2. Nos estágios iniciais da infecção, células T CD4 e CD8 de animais infectados com L. major expressaram IFN-g após ativação. O tratamento com o inibidor de caspase-8 zIETD (benzoil-oxicarbonil-Ile-Glu(OMe)-Thr-Asp(OMe)-fluorometilcetona) durante a estimulação de células T reduziu a proporção de células T CD8 e a expressão de IFN-g por células T CD4 e CD8. Concluimos que a atividade não apoptótica de caspase-8 pode ser necessária para o estabelecimento da imunidade mediada por células T durante a infecção por L. major.

Palavras-chave: apoptose, caspase-8, células T CD8, IFN-gama, Leishmanioses.

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-g, 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, activation-induced cell death (AICD) occurs upon induction ofFas 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-cellapoptosis and control of cytokine responses in draininglymph nodes upon L. major infection. Activation-induced 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-g 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.

MATERIALS AND METHODS

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 ×10⁶ 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.

CELL SUSPENSIONS AND CULTURES

Cells were obtained from draining (popliteal) lymphnodes from infected mice or from a pool of inguinal and popliteal lymph nodes from normal mice. Cells were re-suspended in DMEM (Invitrogen Life Technologies), supplemented with 2 mM glutamine, 5 ×10^-5 M 2-ME, 10 µg/mL gentamicin, 1 mM sodium pyruvate, 0.1 mM MEM nonessential amino acids, and 10 mM HEPES plus 10% FBS (Invitrogen Life Technologies). Cells (1 ×10⁶/0.5mL) were cultured in duplicate in medium only or stimulated with 10 µg/mL plate-bound anti-CD3 (mAb 2C11; BD Pharmingen) in 48-well vessels. Cultures were set at 37^°C and 7% CO in a humid atmosphere for 24 h. In some experiments, cells were activated with anti-CD3 and incubated with 10 µg/ mL anti-FasL (clone MFL3) or IgG control mAb (BD Pharmingen), or with 40 µM of caspase-8 inhibitor zIETD (zIETD-fmk: benzyl-oxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone), or caspase-9 inhibitor zLEHD (zLEHD-fmk: benzyl-oxycarbonyl-Leu-Glu(OMe)-His-Asp(OMe)-fmk) from Enzyme System Products, or 0.4% DMSO as stock diluent control. Apoptosis was evaluated in T cell subsets by flow cytometry as described below.

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 FITC-labeled 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).

INTRACELLULAR CYTOKINES

Lymph node cells (1 ×10⁶/0.5 mL) obtained from L. major-infected mice (2 wk upon infection) were cultured in 48 well plates with plate bound anti-CD3 (10 µg/mL) in the presence or absence of IL-4 (1 ng/mL), zIETD (40 µM) or 0.4% DMSO. Upon 72 h, PMA (10 ng/mL) and ionomycin (0.5 µg/mL) from Sigma were added to cultures. Some cultures were left without further stimulation. After 1 h, brefeldin A (10 µg/mL, Sigma) was added to all cells. Cells were collected upon 3 h, counted, stained, and analyzed by flow cytometry. For surface markers, cells were treated with allophycocyanin-labeled anti-CD8 or anti-CD4, washed, fixed and stained with PE-anti-IL-10 and FITC-anti-IFNg.

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 (^*).

RESULTS

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 expression of CD44 and staining with 7-AAD were assessed as markers of activation and apoptosis in T cells, respectively. We observed increased activation andapoptosis 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 pathway, 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 cytometry 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-g, but not IL-10 (Fig. 4C and D). Both CD4 and CD8 T cells expressed IFN-g (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-g 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) and L. major (Huang 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-g in T cells from L. major-infected mice. Similarly, CD8 T cells have defective survival and IFN-g 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-g 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-g 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-g 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-kB activation and IL-2 secretion (Silva et al. 2005, Su et al. 2005). In agreement with this idea, defective NF-kB activation affects negatively both proliferation and the production of IFN-g 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.

ACKNOWLEDGMENTS

We thank George A. DosReis (IBCCF-UFRJ) for helpful suggestions. This investigation received financial support from the UNICEF/UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases (TDR) (grant A60281), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Institute for Investigation in Immunology (iii), Millennium Institutes, Brazilian Ministry of Science and Technology. W.F.P. receives an MSc fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).M.F.L. is a research fellow at CNPq, Brazil.

Manuscript received on July 11, 2007; accepted for publication on August 15, 2007; presented by ROGERIO MENEGHINI

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Publication Dates

• Publication in this collection
  10 Mar 2008
• Date of issue
  Mar 2008

History

• Accepted
  15 Aug 2007
• Received
  11 July 2007