Caspase-8 Activity Prevents Type 2 Cytokine Responses and Is Required for Protective T Cell-Mediated Immunity against Trypanosoma cruzi Infection

Abstract

During Trypanosoma cruzi infection, T cells up-regulate caspase-8 activity. To assess the role of caspase-8 in T cell-mediated immunity, we investigated the effects of caspase-8 inhibition on T cells in viral FLIP (v-FLIP) transgenic mice. Compared with wild-type controls, increased parasitemia was observed in v-FLIP mice infected with T. cruzi. There was a profound decrease in expansion of both CD4 and CD8 T cell subsets in the spleens of infected v-FLIP mice. We did not find differences in activation ratios of T cells from transgenic or wild-type infected mice. However, the numbers of memory/activated CD4 and CD8 T cells were markedly reduced in v-FLIP mice, possibly due to defective survival. We also found decreased production of IL-2 and increased secretion of type 2 cytokines, IL-4 and IL-10, which could enhance susceptibility to infection. Similar, but less pronounced, alterations were observed in mice treated with the caspase-8 inhibitor, zIETD. Furthermore, blockade of caspase-8 by zIETD in vitro mimicked the effects observed on T. cruzi infection in vivo, affecting the generation of activated/memory T cells and T cell cytokine production. Caspase-8 is also required for NF-κB signaling upon T cell activation. Blockade of caspase-8 by either v-FLIP expression or treatment with zIETD peptide decreased NF-κB responses to TCR:CD3 engagement in T cell cultures. These results suggest a critical role for caspase-8 in the establishment of T cell memory, cell signaling, and regulation of cytokine responses during protozoan infection.

Inhibition of caspase-8 activity promotes protective Th1- and Th2-mediated immunity toLeishmania majorinfection

“…We have reported previously that CD4 T cells from vFLIP or FasL-mutant gld mice also secrete increased amounts of IL-4 and IL-10 upon infection with Trypanosoma cruzi [35,45]. Although alterations in cytokine expression correlated with increased susceptibility to T. cruzi infection [35,45], resolution of L. major infection in vFLIP mice was not negatively affected by increased IL-4 production.…”

“…IL-13 and IFN-␥ cytokines were also increased in response to L. ma- jor antigens. Although possible negative effects of caspase-8 inhibition on TCR signaling, T cell proliferation, and IFN-␥ production should be taken into account [31,35,42], these effects may have been overcome by rescuing of CD4 ϩ Th1 cells from Fas-induced apoptosis in infected vFLIP mice, as observed previously in FasL/Fas-defective mice [26,28]. Similarly, inhibition of caspase-8 can favor Th2 responses in vFLIP mice by improving survival of CD4 T cells, as supported by comparable effects of blocking FasL and caspase-8 activity in vitro.…”

“…For evaluation of apoptosis, T cells (1ϫ10 6 /0.5 ml) from normal and infected mice were cultured in triplicates and stimulated with 10 g/ml plate-bound anti-CD3 (mAb 2C11; BD PharMingen, San Diego, CA, USA) in the presence or absence of 10 g/ml anti-FasL mAb (MFL3 clone, hamster IgG1), anti-TRAIL (N2B2, rat IgG2a), anti-TNF-␣ (G281-2626, rat IgG1), or respective control IgG mAb (all from BD PharMingen) or in the presence of 40 M caspase-8 inhibitor zIETD-fmk, caspase-9 inhibitor zLEHD-fmk (Enzyme Systems Products, Livermore, CA, USA), or control diluent (0.4% DMSO) [35] in 48-well vessels for 24 h. Apoptosis was evaluated in gated CD4 ϩ cells by flow cytometry as described below. All cultures were set at 37°C and 7% CO 2 in a humid atmosphere.…”

“…It is noteworthy, however, that caspase-8 activity is also required for other aspects of T cell activation and development of immune responses to viral and parasite infections [13, 31, 34 -37]. As we found previously that vFLIP expression negatively affects the production of cytokines by CD8 T cells and CD8 T cell-mediated immunity [31,35], we chose the L. major infection model to explore the effects of caspase-8 inhibition on CD4 T cells. Despite expressing increased Th2 cytokines along with a robust Th1 response, vFLIP mice exerted a better control over L. major infection, with reduced lesions and parasite loads, than WT mice.…”

Monitoring the caspase cascade in single apoptotic cells using a three-color fluorescent protein substrate

“…Most apoptosis depending on the caspase cascade, as well as the cascade the process itself, is obstructed by caspase-inhibitors [11][12][13]. The current method to monitor caspase activities includes the use of caspase substrates and inhibitors [14][15][16][17][18][19][20][21]. Previously, we constructed tandem fused fluorescent proteins with two colors for measurement of caspase-3 activity [22], and three colors for measurement of caspase-3 and caspase-9 activities [23] by using FCCS.…”