Reduced myocarditis following Coxsackievirus infection in cellular FLICE inhibitory protein – long form-transgenic mice

Abstract

Cellular FLICE inhibitory protein – long form (c-FLIP_L) is a caspase-defective homologue of caspase-8 that blocks apoptosis by death receptors. c-FLIP_L expression in T cells can also augment activation of the mitogen-activated protein kinase, extracellular signal-related kinase, as well as nuclear factor-κB. This contributes to increased production of interleukin-2 and CD25, resulting in hyperproliferation of T cells from c-FLIP_L-transgenic mice. c-FLIP also heterodimerizes with and activates caspase-8, resulting in increased death of T cells and a selection of a T helper 2 cytokine profile. The effects of c-FLIP on cytolytic function of CD8⁺ T cells have not been examined previously. We studied the cytolytic capacity of T cells from c-FLIP_L-transgenic mice using an antigen-specific system, as well as the consequences during a viral immune response to Coxsackievirus B3 (CVB3). The increased T-cell receptor (TCR) signalling due to c-FLIP did not alter the cytolytic machinery but did reduce cytotoxicity because of decreased surface expression of TCR and CD8. It also produced a Tc2 cytokine profile. These effects of c-FLIP collectively served to diminish the severity of CVB3-induced myocarditis.

Introduction

Death receptors typified by tumour necrosis factor receptor-1 (TNFR1) and Fas mediate apoptosis in a wide array of cell types through the ligand-induced association of adaptor proteins that in turn recruit a series of cysteine-requiring aspartate-specific proteases known as caspases.¹ In the case of Fas, oligomerization via FasL promotes the binding of Fas-associating death domain (FADD) to the death domain of Fas.² This allows the association of caspase-8 with FADD via their mutual death effector domains (DED), forming the death-inducing signal complex (DISC). Pro-caspase-8 can then undergo autocatalytic cleavage at critical aspartate residues to form fully active caspase-8. The resulting protease cascade cleaves and activates caspase-3 leading to apoptosis.³

Caspase-8 is also critical for activation of T cells^4,5 yet it remains unclear how procaspase-8 is activated following T-cell receptor (TCR) stimulation. Recent evidence suggests that the death receptor inhibitor, cellular FLICE inhibitory protein (c-FLIP), is an activator of caspase-8 following TCR stimulation.^6,7 FLIP was originally identified as a family of six viral inhibitors (v-FLIPs) that are present in several γ-herpes viruses and molluscipox virus, and contain two DED that can bind to FADD and thus compete with caspase-8 binding at death receptors.⁸ v-FLIPs block the early signalling events of a variety of death receptors including Fas, TNFR1, transgenic adenocarcinoma of the mouse prostate (TRAMP) (DR-3, Apo-3), and tumour necrosis factor related apoptosis inducing ligand receptor (TRAILR) (DR4, DR5).⁸ A mammalian cellular homologue (c-FLIP) of v-FLIP was later identified from an expressed sequence tag database and cloned.⁹ c-FLIP is strongly expressed in heart, skeletal muscle, and lymphoid tissues, and exhibits close overall structural homology to caspase-8.^9,10 As with v-FLIP, the full-length 55 000 MW long form of c-FLIP (c-FLIP_L) contains two DED that interact with the DED of FADD.¹¹ c-FLIP_L contains a C-terminal inactive caspase-like domain, but retains a caspase-8 cleavage site at Asp374.¹¹ As such, c-FLIP_L acts as a caspase-8 substrate trap in that it is cleaved by caspase-8 upon its recruitment to the Fas/FADD complex.⁹ c-FLIP-deficient mice are embryonic lethal because of a cardiac malformation¹² highlighting that c-FLIP is likely important for cell processes other than merely protection from Fas-induced death.

In addition to its ability to inhibit caspase-8 recruitment to the DISC, c-FLIP_L can heterodimerize with and activate caspase-8 through their mutual N-terminal DED and an activation loop in the C-terminal caspase-like domain of c-FLIP_L.^6,7 c-FLIP is then rapidly cleaved to p43FLIP, which allows it to recruit receptor interacting protein 1 (RIP1) and TNF receptor associating factor (TRAF)1/2, which promotes activation of the nuclear factor (NF)-κB pathway.^13–15 c-FLIP also associates with Raf1 and augments activation of the mitogen-activated protein (MAP) kinase extracellular signal-related kinase (ERK).¹³ Consequently, increased expression of c-FLIP in T cells results in enhanced production of interleukin-2 (IL-2), expression of CD25, and proliferation.^15,16 Conversely, T cells lacking c-FLIP manifest a profound defect in proliferation.^17,18 The ability of c-FLIP_L to both promote ERK and NF-κB activation, combined with its ability to activate caspase-8, results in a complex pattern of both increased proliferation as well as accelerated cell death, particularly of the CD8⁺ subset.^15,19 Thus, the levels of c-FLIP in T cells can be pivotal to the outcome of an immune response. In the CD4⁺ subset, increased c-FLIP expression manifests as a T helper 2 (Th2) cytokine bias, promoting increased susceptibility to the development of allergic airway hypersensitivity.²⁰ However, the effect on cytolytic activity of CD8⁺ T cells has not been examined. Hence, in the current study we explored the effect of increased c-FLIP expression in T cells on the CD8-dependent myocardial inflammation resulting from Coxsackievirus B3 (CVB3) infection²¹ and cytolysis toward cardiac endothelial cells. We find that the augmented activation of c-FLIP_L-transgenic (Tg) CD8⁺ T cells results in substantial down-regulation of surface TCR and CD8, and hence reduced cytotoxicity, as well as a Tc2 cytokine phenotype, collectively resulting in decreased myocardial damage.

Materials and methods

Mice

c-FLIP_L was expressed transgenically in the T-cell compartment as previously reported.¹⁶ Briefly, amino acid sequence DYKDDDDK (FLAG)-tagged mouse FLIP_L cDNA was inserted into a target vector containing the β-globin promoter and a downstream human CD2 locus enhancer element. Transgenic mice were screened by PCR of ear DNA using the following primers:

5′ Primer: (5′- GGAGCCAGGGCTGGGCATAAAA-3′)

3′ Primer: (5′-GACTCACCCTGAAGTTCTCAGGATCC-3′).

Immunoblot using anti-FLIP monoclonal antibody (mAb; Dave-2, Apotech, Lausen, Switzerland) further confirmed expression of the transgene. The c-FLIP_L-Tg mouse strain has been backcrossed to C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) for nine generations.

OT-I mice bear a transgenic TCR that recognize chicken ovalbumin (OVA) peptide 257–264 (SIINFEKL) restricted to class I major histocompatibility complex (MHC), K^b, and were kindly provided by Drs Francis Carbone and Michael Bevan.²² OT-I mice were maintained by breeding TCR Tg male mice to normal C57BL/6 females. Offspring were screened for the clonotype TCR using anti-Vα2 mAb. The animal facility is AALAC-approved, and protocols were approved by the UVM IACUC. Original breeding pairs of C57BL/6^+/+ were obtained from the Jackson Laboratories.

Virus infection

Animals were infected by intraperitoneal (i.p.) injection of 0·5 ml phosphate-buffered saline (PBS) containing 10⁵ plaque-forming units (PFU) of CVB3 (H3 strain), derived from Cos cells transfected with the infectious viral cDNA.²³ For virus titration, hearts were divided in half. One half was placed in 10% formalin for histology. The other half was homogenized in 0·9 ml RPMI-1640 containing 100 U/ml penicillin, 100 µg/ml streptomycin, and 5% fetal bovine serum (FBS). Cellular debris was removed by centrifugation at 1045 g for 10 min. The supernatant was titred by the plaque-forming assay on HeLa cell monolayers as described previously.²⁴

Lymphocyte isolation

Spleens were removed and pressed through fine mesh screens into RPMI-1640 medium. Cell debris was allowed to settle and the supernatant was centrifuged on Histopaque-1077 (Sigma-Aldrich, St. Louis, MO). Cells at the interface were removed, washed once and incubated on nylon wool for 30 min at 37°. The non-adherent cells were retrieved. For isolation of purified CD8⁺ T cells, the nylon wool purified cells were incubated with anti-CD4 (clone GK1.5), anti-IA/IE (clone 2G9), and anti-γδ TCR (clone GL3) mAbs. Cells were then washed, incubated with immunoglobulin G (IgG) mouse anti-hamster IgG (clone HIG-29), followed by rat anti-mouse IgG and then anti-rat IgG-conjugated magnetic particles (PerSeptive Biosystems, Framingham, MA). The sample was then placed on a magnet to remove labelled cells. The residual cells in the suspension were 91% CD8⁺ by flow cytometry using clone 53-6.7. All antibodies were from BD-PharMingen (San Diego, CA).

Lymphocyte culture

Ovalbumin peptide 257–264 (SIINFEKL) (OVAp) was produced at Macromolecular Resources (Colorado State University, Fort Collins, CO). OT-I and OT-I/c-FLIP_L-Tg lymph node T cells were cultured in culture medium (RPMI-1640, 2·5 mg/ml glucose (Sigma), 10 mg/ml folate (Invitrogen, Carlsbad, CA), 110 µg/ml pyruvate (Invitrogen), 5 × 10⁻⁵ m 2-mercaptoethanol (2-ME; Sigma), 292·3 µg/ml glutamine (Invitrogen), 100 U/ml penicillin–streptomycin (Invitrogen), and 5% fetal calf serum) supplemented with recombinant human IL-2 (50 U/ml, Cetus, Emeryville, CA) and OVAp (10⁻⁷ m). Cells were expanded over 5 days and used as effector cytolytic T cells.

Flow cytometry

Details for intracellular cytokine staining have been published previously.²⁵ Briefly, 10⁶ lymphocytes were cultured for 4 hr in RPMI-1640 medium containing 5% fetal bovine serum (FBS), 10 µg/ml of brefeldin A (BFA), 50 ng/ml phorbol myristate acetate (PMA), and 500 ng/ml ionomycin (Sigma). After culture, the cells were washed in PBS containing 1% bovine serum albumin (BSA, Sigma) containing BFA, incubated with 1 : 100 dilution of Fc-Block, fluoroscein isothiocyanate (FITC)–anti-CD8 (clone 53-6.7) and PerCP-Cy5.5–anti-CD4 (clone RM4-5) (all from BD PharMingen) for 20 min at 4°, washed once with PBS–BSA–BFA, then fixed in 2% paraformaldehyde for 10 min. The cells were resuspended in PBS–BSA containing 0·5% saponin, Fc-Block and 1 : 100 dilutions of PE anti-IFN-γ (clone XMG1·2, BD PharMingen). Matching fluorochrome-conjugated rat IgG1 was used as isotype control. The cells were incubated for 30 min on ice, washed once in PBS–BSA–saponin and once in PBS–BSA, then resuspended in 2% paraformaldehyde and analysed using a BD LSR II (BD Immunocytometry, San Jose, CA).

Histology

Hearts were removed, fixed in 10% buffered formalin, paraffin embedded, sectioned, and stained with haematoxylin and eosin. Sections were then scored blindly on a 0–4 scale where 0 represents no myocarditis, 1 represents 1–10 inflammatory foci/heart section, 2 represents 11–20 foci/section, 3 represents 21–30 foci/section and 4 represents > 40 foci/section.

Virus neutralizing antibody

Plasma was serially diluted 1 : 4 in RPMI-1640 medium containing antibiotics and 5% FBS and incubated with 100 PFU CVB3 for 30 min in a total volume of 100 µl. The virus–plasma mixture was transferred to 96-well tissue culture plates containing confluent monolayers of HeLa cells and the plates were incubated for at 37° for 24 hr in a humidified 5% CO₂ incubator. The monolayers were fixed for 15 min with 10% buffered formalin then stained with 4% crystal violet. The wells were washed thoroughly with water and the plates were read at 630 nm on a BioTek EL808 plate reader with KC4 software (BioTek Instruments, Winooski VT). Antibody titre was the antibody dilution at which 50% of the monolayer was killed.

Statistics

Histology and virus titres were statistically evaluated using Wilcoxon ranked score. Cytotoxicity assays were evaluated by Student's t-test. Mortality was evaluated by Chi-squared analysis.

Cell-mediated cytotoxicity assay

For cytotoxicity by effector OT-I cells, EL-4 target cells (H-2^b) were labelled with 30 µl/ml of 5 mCi

CrSO₄ (New England Nuclear, Wellesley, MA) for 1 h at 37°. Cells were washed five times with RPMI + 5% BCS. OT-I effector T cells were serially diluted in a separate 96-well plate followed by the addition of labelled EL-4 cells (1 × 10⁴/well) and OVAp at 10⁻¹⁰ m in a final volume of 200 µl. In some assays, concanavalin A (Con A, 1 µg/ml) was added to promote cell contact and test the inherent cytolytic potential of T cells in the absence of specific recognition of target cells.²⁶ After 4 hr incubation at 37° 100 µl of supernatant was removed and ⁵¹Cr release (counts per minute, c.p.m.) determined using a gamma counter. HCl (3·0 N) was diluted 1 : 1 into select wells to determine maximal ⁵¹Cr release. Percent specific lysis was calculated as follows: [(experimental c.p.m. − spontaneous release c.p.m.)/(maximum release c.p.m. − spontaneous release c.p.m.)] × 100.

Cell mediated cytotoxicity by CD8⁺ effector T cells from CVB3-infected mice for cardiac myocytes has been described in detail previously.²⁷ Cardiac endothelial cells were isolated from the hearts of 6 week-old C57BL/6 mice. Hearts were perfused with PBS, minced finely and digested with 0·25% pancreatin (Life Technologies, Gaithersburg, MD) and 0·4% collagenase (Worthington Biochemical, Freehold, NJ). The single cell suspension was washed twice with RPMI-1640 medium containing antibiotics and 5% FBS, then incubated on plastic Petri dishes at 37° for 1 hr. The non-adherent cells were removed by washing. The adherent cells were trypsinized, washed, counted by trypan blue exclusion, resuspended to 1 × 10⁶ cells/ml, and 100 µl (10⁵ cells/well) dispensed into 96-well tissue culture well plates. After overnight incubation, these target cells were labelled with 100 µCi/ml

CrO₄ (MP Biomedical, Irvine CA) at 37° for 2 hr, washed three times, then incubated at a 30 : 1 ratio with purified CD8⁺ T cells from the spleen at 37° for 18 hr. Supernatants were removed and counted in a Packard Gamma Counter (Packard Instruments, Downers Grove, IL). Adherent cells in each well were lysed using 0·6 N HCl and counted. Percent ⁵¹Cr release was calculated as: [c.p.m. in supernatant/(c.p.m. in supernatant and c.p.m. in pellet)] × 100.

Perforin PCR

Lymphocytes (5 × 10⁶) were lysed in Ultraspec™ RNA reagent (Biotecx Laboratories, Houston, TX) and RNA was prepared according to the manufacturer's directions. Oligo(dT) priming and reverse transcriptase was used to prepare cDNA from RNA samples. Polymerase chain reaction (PCR) amplifications were performed as follows: 94° × 1 min, 55° × 1 min, 72° × 1 min (35 cycles). Primers used for amplification of perforin have been described previously.²⁸ PCR reactions were separated on 1·5% agarose gels and products visualized by ethidium bromide staining. Densitometry was quantitated using a digital camera and Alpha Imager software.

Results

Activated c-FLIP_L-Tg CD8⁺ T cells manifest decreased surface TCR and CD8 and decreased cytolytic potential

c-FLIP can associate with Raf1 to promote activation of ERK, and also associate with TRAF1/2 and RIP1 to augment activation of NF-κB.¹³ As these pathways can affect many effector functions of T cells, we examined the cytolytic capacity of c-FLIP_L-Tg T cells following activation both in vitro and in vivo. In the first case, lymph node or spleen cells from OT-I and OT-I/c-FLIP_L-Tg mice were activated in vitro with OVAp (10⁻⁷ m) plus IL-2 (50 U/ml) for 5 days (Fig. 1a). In the second situation, OT-I and OT-I c-FLIP_L-Tg mice received two injections of OVAp (100 µl of 100 µm) 24 hr apart, and lymph node and liver lymphocytes harvested 48 hr after the last injection (Fig. 1b). Purified CD8⁺ cells from these sites were used from both sources as effector cytolytic T cells (CTL), and targets consisted of ⁵¹Cr-labelled syngeneic EL4 cells pulsed with 10⁻¹⁰ m OVAp. Cytolytic activity of OT-I/c-FLIP_L-Tg CD8⁺ T cells was considerably diminished on a per-cell basis compared to OT-I T cells (Fig. 1a, b). Activated CD8⁺ T cells can migrate to the liver²⁹ and could selectively deplete lymph nodes of cytolytic capacity. This was not the case, however, as liver CD8⁺ lymphocytes from OT-I/c-FLIP_L-Tg mice also manifested reduced cytolytic potential (Fig. 1b).

Figure 1.

Low cytolytic capacity of OT-I/c-FLIP_L-Tg CD8⁺ T cells. CD8⁺ T cells from OT-I and OT-I/c-FLIP_L-Tg mice were activated (a) in vitro with OVAp (10⁻⁷ m) plus IL-2 for 5 days or (b) in vivo with two injections of OVAp (100 µl of 100 µm each, 24 hr apart). CD8⁺ T cells were then purified and used as cytolytic effector cells with ⁵¹Cr-labelled syngeneic targets of EL4 or C57BL/6 B-cell blasts pulsed with 10^–10 m OVAp. After 4 hr, ⁵¹Cr release was quantitated and percent maximum lysis (HCl) was determined. Results represent mean ± SD of three separate experiments.

The two principal mechanisms of in vitro cytotoxicity are mediated by perforin and Fas-Ligand (FasL).³⁰ Expression of surface FasL was found to be as high, if not higher, in OT-I/c-FLIP_L-Tg CD8⁺ T cells than in OT-I T cells (Fig. 2a). In addition, levels of perforin expression in CD8⁺ T cells were also similar between the two groups of effector T cells (Fig. 2b). Hence, differences in FasL or perforin expression could not explain the diminished cytotoxicity of OT-I/c-FLIP_L-Tg CD8⁺ T cells.

Figure 2.

FasL and perforin expression are not reduced in OT-I/c-FLIP_L-Tg T cells. (a) Surface FasL was determined by flow cytometry comparing day 5 effector OT-I cells (left panel) with OT-I/c-FLIP_L-Tg cells (right). Solid line indicates the levels of FasL staining compared to dotted line showing isotype control antibody. Number insert represents percent positive cells. (b) Perforin mRNA levels assessed by PCR compared to hprt gene expression. RNA was obtained from the same effector T cells as in (a), and then cDNA was made and titred in PCR reactions as shown.

In contrast to perforin and FasL expression, both surface TCR and CD8 were decreased on OT-I/c-FLIP_L-Tg T cells compared to OT-I cells following OVAp activation (Fig. 3a). These differences were not apparent at the mRNA level (data not shown) and thus likely reflect surface down-modulation. This would be consistent with the known regulation of surface TCR and CD8 by ERK and NF-κB,^31,32 as well as with the ability of c-FLIP_L to augment signalling of these two pathways.¹³ Surface TCR and CD8 are critical recognition structures for mediating specific cytolysis.³³ To bypass the need for these recognition molecules and directly test the cytolytic machinery, lectins such as Con A can directly bind effector CTL to target cells.³³ This considerably augmented the lytic capacity of OT-I/c-FLIP_L-Tg T cells and to a much lesser extent OT-I effector T cells (Fig. 3b). This supports the view that the intrinsic lytic capacity of c-FLIP_L-Tg CD8⁺ T cells was fully intact.

Figure 3.

Decreased surface CD8 and TCR expression by OT-I/c-FLIP_L-Tg T cells. (a) Surface expression of CD8 and TCR Vα2 determined by flow cytometry comparing day 5 effector OT-I cells (dotted line) with OT-I/c-FLIP_L-Tg cells (solid line). Number inserts represent mean fluorescence intensity. (b) Cytolytic activity of day 5 OT-I and OT-I/c-FLIP_L-Tg CD8⁺ T cells toward OVAp-pulsed EL4 cells in the absence or presence of Con A (1 µg/ml).

Decreased myocardial inflammation in c-FLIP_L-Tg mice infected with Coxsackievirus B3

The above findings were applied to an in vivo CD8⁺ T-cell dependent inflammatory process, Coxsackievirus B3 (CVB3)-induced myocarditis. In C57BL/6 male mice, CVB3-induced myocarditis is an autoimmune response to the virus that is mediated by CD8⁺ T cells.²¹ The findings shown in Figs 4 and 5 are from one of four replicate experiments. In this study a group of 16 B6 c-FLIP_L-Tg male mice and 23 B6^+/+ normal littermate control male mice were infected with 10⁵ PFU of CVB3. Animal mortality was 11/23 for B6^+/+ mice (45%) and 6/16 (35%) for B6 c-FLIP_L-Tg mice (Fig. 4), demonstrating a slight though not statistically significant decrease in mortality in c-FLIP_L-Tg animals. However, the cause of death following CVB3 infection is multifactorial and not caused merely by cardiac inflammation. Thus, surviving mice were examined more closely to evaluate the degree of myocarditis. Mice surviving to 7 days after infection (12 wild-type and 10 c-FLIP_L-Tg mice) were killed. Analysis of hearts revealed a prominent inflammatory infiltrate in the wild-type mice, whereas hearts from the c-FLIP_L-Tg mice showed significantly less mononuclear infiltrate. An example of cardiac histology is shown in Fig. 5(a) and summarized for all mice in Fig. 5(b). In addition, viral burden was somewhat reduced in c-FLIP_L-Tg mice in the experiment shown, although this difference was not statistically significant over the four experiments (Fig. 6a). Virus neutralizing antibody titres were evaluated in the plasma of surviving mice (Fig. 6b) and showed significantly greater levels in c-FLIP_L-Tg mice compared to wild-type controls. The lower neutralizing antibody titres in wild-type animals would likely explain their higher virus titres

Figure 4.

Equivalent mortality of wild-type and c-FLIP_L-Tg mice following CVB3 infection. Male C57BL/6 (B6) wild-type littermate controls (23 mice) or B6 c-FLIP_L-Tg (16 mice) (age 8 weeks) were infected with 10⁵ colony-forming units of CVB3. One week later 12 wild-type and 10 c-FLIP_L-Tg mice survived.

Figure 5.

Decreased myocarditis in CVB3-infected c-FLIP_L-Tg mice. (a) Histology of male B6 wild-type and B6 c-FLIP_L-Tg mice infected i.p. 7 days earlier with 10⁵ PFU CVB3. Hearts were divided in half with one half formalin fixed, paraffin embedded, sectioned, and stained with haematoxylin and eosin. The other half was used to measure viral titres. Cardiac sections were scored for inflammation on a 0–4 scale and showed a moderate mononuclear inflammatory infiltrate in the B6 wild-type heart (left panel) but little in the c-FLIP_L-Tg heart (right panel). Arrows indicate cardiac inflammation. (b) Composite cardiac inflammation score. These findings were similar in three separate experiments.

Figure 6.

Cardiac virus titres and viral antibody responses. (a) Heart sections from CVB3-infected mice were homogenized and homogenates titred on HeLa cell monolayers and lysis measured. Shown is one of four experiments in which the composite titres were not significantly different. (b) Plasma from the same mice was serially diluted and evaluated for neutralization of 100 PFU CVB3. The titre represents the plasma dilution blocking 50% virus lysis of HeLa cells.

We have previously observed that the proportion of CD8⁺ T cells is decreased in c-FLIP_L-Tg mice by 30–50%.¹⁶ The reason for this is an augmented level of caspase activity caused by increased c-FLIP_L expression that manifests particularly in the CD8⁺ subset with increased cell death.¹⁹ Thus, phenotypic analysis of spleen cells from CVB3-infected mice revealed that the proportion of CD8⁺ cells was decreased about 50% in c-FLIP_L-Tg mice while the proportion of CD4⁺ cells was slightly increased (Fig. 7a). In addition, the intracellular cytokine profile of c-FLIP_L-Tg splenocytes demonstrated a reduction in interferon-γ (IFN-γ) production compared to wild-type splenocytes (Fig. 7b). By contrast, the levels of IL-4 were equivalent or slightly increased in c-FLIP_L-Tg T cells (Fig. 7b). These data are consistent with earlier studies of c-FLIP_L mice showing a Th2 bias of both CD4⁺ and CD8⁺ subsets following in vitro stimulation with anti-CD3/CD28.²⁰

Figure 7.

Lymphocytes from CVB3-infected c-FLIP_L-Tg mice manifest fewer CD8⁺ T cells and diminished production of IFN-γ. (a) Spleen cells from CVB3-infected wild-type or c-FLIP_L-Tg mice were evaluated by flow cytometry for the percent positive CD4⁺ and CD8⁺ cells. (b) Flow cytometric analysis of peripheral blood mononuclear cells (PBMC), spleen cells, and mesenteric lymph node cells stimulated with PMA plus ionomycin for 4 h in the presence of brefeldin A, then labelled for expression of surface CD8 and intracellular IFN-γ or IL-4. (c) Cytolytic activity of purified CD8⁺ T cells from uninfected wild-type, CVB3-infected wild-type, and CVB3-infected c-FLIP_L-Tg mice toward ⁵¹Cr-labelled cardiac target cells at a 30 : 1 effector : target ratio. Shown is the percentage maximal lysis compared to HCl controls.

The myocarditis in male C57BL/6 mice is caused by CD8⁺-mediated cytotoxicity of cardiocytes rather than death of myocytes induced directly by the virus.^21,34 We therefore examined the cytolytic potential of purified CD8⁺ splenocytes toward ⁵¹Cr-labelled cardiac myocytes in vitro. As shown in Fig. 7(c), purified CD8⁺ spleen cells from c-FLIP_L-Tg mice manifested 60% less lytic activity to cardiocyte targets on a per cell basis than CD8⁺ cells from infected wild-type animals (effector : target ratio of 30 : 1). These findings are in agreement with the decreased cytolytic activity of CD8⁺ T cells from OT-I/c-FLIP_L-Tg mice, and confirm that the cytolytic potential of c-FLIP_L-Tg CD8⁺ cells is diminished.

Discussion

The current findings reveal that the levels of c-FLIP in T cells can profoundly alter the immune response to infectious pathogens such as CVB3. c-FLIP induced a decrease in CD8⁺ cytolytic T-cell activity, as well as a diminished IFN-γ response and slightly increased IL-4 production. A Th1 cytokine profile has been demonstrated previously to promote myocardial inflammation during CVB3 infection.³⁵ In addition, a Th2 cytokine bias has been observed previously in CD4⁺ and CD8⁺ T cells from c-FLIP_L-Tg mice.²⁰ The diminished cytolytic activity was caused by decreased surface CD8 and TCR. This likely resulted from the known ability of c-FLIP to augment activation of ERK and NF-κB,¹³ both of which are linked to down-modulation of surface CD8 and TCR.^31,32 Collectively, these parameters likely act synergistically to reduce myocardial inflammation in c-FLIP_L-Tg mice.

During the past few years two unanticipated functions of c-FLIP_L have been defined. The first is that c-FLIP_L can actually activate caspase-8.⁶ This initially seems paradoxical to the traditional view that c-FLIP_L blocks Fas-induced death. However, structural modelling has revealed that c-FLIP_L and pro-caspase-8 can directly heterodimerize, even in the absence of Fas signalling or cell death in effector T cells.^6,7 In distinction to downstream effector caspases that require cleavage to become activated, heterodimerized c-FLIP_L/caspase-8 results in activation of caspase-8 in its full-length non-cleaved form because of an activation loop that overlaps and opens the active site of caspase-8, without actually cleaving caspase-8.⁶ An additional point is that there is a caspase-8 substrate sequence within the activation loop of c-FLIP_L at Asp374. As this sits directly over the enzymatically active site of caspase-8, c-FLIP_L can be cleaved at Asp374, resulting in a p43FLIP fragment with decreased affinity for the heterodimeric complex. Although p43FLIP cannot activate caspase-8, it is able to bind TRAF2 and RIP1 more avidly than full-length c-FLIP_L and promote activation of NF-κB.^14,15 p43FLIP may also serve to limit caspase-8 activation by c-FLIP_L.

The second new function of c-FLIP_L derives from studies in cell lines showing that c-FLIP is able to bind Raf-1, TRAF-1/2, and RIP1, which activate, respectively, the MAP kinase ERK and NF-κB pathways.¹³ As a result, increased expression of c-FLIP_L in Jurkat T cells resulted in increased IL-2 production upon TCR ligation¹³ and in hyperproliferation of both CD4⁺ and CD8⁺ T cells from c-FLIP_L-Tg mice.^15,16,20 Several tumour cell lines³⁶ and fibroblasts³⁷ also contain high levels of c-FLIP and are not only resistant to Fas-induced death, they actually hyperproliferate with Fas ligation. In addition, ligation of Fas on dendritic cells, which also express very high levels of c-FLIP, actually promotes up-regulation of CD80, CD86, MHC class II, and IL-12 production³⁸ suggesting a role for Fas in positive signalling of dendritic cells. These collective findings suggest that c-FLIP may have a dual function of not only inhibiting Fas-induced cell death, but also to actively promote cell growth and/or differentiation.

Given the earlier observations that increased expression of c-FLIP_L in T cells resulted in both hyperproliferation and caspase-dependent increased cell death^15,19 it was not clear a priori how this would manifest during the immune response to an active viral infection. CVB3 infection causes a secondary inflammatory response in the heart that is not caused by direct viral cytopathic effects on cardiac myocytes, but rather, independently via both IFN-γ expression³⁵ and cytolytic CD8⁺ T cells.^21,39 Given these features, several factors likely contributed to the decreased myocarditis observed in c-FLIP_L-Tg mice. The first is the decreased survival of CD8⁺ T cells because of augmented caspase activity of c-FLIP_L-Tg T cells. Thus, despite the augmented proliferation of these T cells, they likely undergo apoptosis before they can significantly infiltrate the myocardium. Second, the decreased IFN-γ expression by c-FLIP_L-Tg CD8⁺ T cells following CVB3 infection is consistent with the known Th2 phenotype of c-FLIP_L-Tg CD4⁺ T cells.²⁰ Third, the decreased cytotolytic activity of c-FLIP_L-Tg CD8⁺ T cells occurs on a per cell basis. This was not the result of decreased expression of either FasL or perforin, both of which were normal or even elevated in c-FLIP_L-Tg CD8⁺ effector T cells. Rather, the explanation would appear to result from decreased surface expression of TCR and CD8 following activation of c-FLIP_L-Tg CD8⁺ T cells. This would be consistent with the ability of Con A to reverse the defect in cytotoxicity through the induced association of effector and target cells. Finally, the augmented neutralizing antiviral antibodies in c-FLIP_L-Tg mice is likely to have resulted from the Th2 phenotype of these mice, and could certainly contribute to the diminished inflammation, although viral titres were not different between the two strains of mice.

An interesting feature of c-FLIP_L-Tg T cells that likely influences the course of CVB3 infection is the cytokine response. Both CD4⁺ and CD8⁺ T cells from c-FLIP_L-Tg mice manifest a Th2 cytokine profile.^15,20 While the mechanism for this bias is currently uncertain, several features of c-FLIP_L-Tg T cells likely lead to this phenotype. First, c-FLIP_L-Tg T cells manifest increased levels of GATA-3, a major transcriptional regulator of Th2 cytokines, and reduced levels of Tbet, a regulator of Th1 cytokines.²⁰ The molecular explanation for this is not presently known, but a likely scenario is that the augmented caspase activity in c-FLIP_L-Tg T cells favours the preferential survival of Th2 cells, because of their higher levels of Bcl-2⁴⁰. c-FLIP_L-Tg T cells also manifest less nuclear factor of activated T cells c2 (NFATc2), because of its cleavage by caspase-3.⁴¹ Because NFATc2^–/– mice also manifest a Th2 bias, the reduced NFATc2 levels in c-FLIP_L-Tg T cells may also contribute to their Th2 bias.⁴² Hence, we feel that the increased caspase activity in c-FLIP_L-Tg T cells is the likely source of the Th2 bias. The Th2 bias of c-FLIP_L-Tg mice contributes to an increased severity of allergic hypersensitivity airway disease.²⁰ From these studies it was less clear whether this Th2 bias would predominate during an infection that drives a Th1 response. The myocarditis following CVB3 infection is highly dependent upon a Th1 cytokine response³⁵ and CVB3 variants that evoke a Th2 response do not cause myocarditis.⁴³ Hence, the cytokine profile of the c-FLIP_L-Tg mice likely synergizes with the decreased cytolytic response to reduce myocardial inflammation with CVB3 infection.

Abbreviations

CVB3

Coxsackievirus B3

DED

death effector domain

FasL

Fas-ligand

c-FLIP_L

cellular FLICE inhibitory protein – long form