IKK antagonizes activation-induced cell death of CD4+ T cells in aged mice via inhibition of JNK activation
Abstract
T cell dysfunction is the primary immunologic abnormality associated with aging. Many age-related defects stem from a decline in CD4+ T cell function. Resistance of aged CD4+ T cells to apoptosis is asso- ciated with autoimmune and infectious diseases. Previous studies suggest that InB kinase (IKK) may be a key player in cell survival via its inhibition of c-Jun N-terminal protein kinase (JNK) activation. How- ever, the role of IKK-mediated JNK inactivation in the age-related apoptosis of T cells is unclear. Here, we report that splenic CD4+ T cells in aged mice are resistant to activation-induced cell death (AICD) induced by anti-CD3 plus IL-2 stimulation. Furthermore, aged CD4+ T cells display increased IKKβ activity that is associated with attenuated JNK activation. The IKKβ-mediated JNK inactivation in aged CD4+ T cells reduces the degradation of c-FLIPL and the interaction of Bad with Bcl-XL, but it increases the affinity of Bad for 14-3-3. Pretreatment of aged CD4+ T cells with a specific IKK inhibitor, PS1145, increases the JNK activity blocked by IKKβ and consequently sensitizes the aged CD4+ T cells to AICD. Our study thus demonstrates that IKK antagonizes the AICD of CD4+ T cells in aged mice via inhibition of JNK activation.
1. Introduction
Aging is a natural and inevitable part of the life process and is accompanied by pleiotropic changes in the immune system that lead to dysfunction of cellular and humoral immune responses (Linton and Dorshkind, 2004). An age-related progressive accumu- lation of cellular damage is associated with increased susceptibility to infectious disease, autoimmune disease, and cancer (Prelog, 2006; Tsukamoto et al., 2009). T lymphocyte apoptosis is essen- tial for the proper function of immune system. T cell receptor (TCR) re-stimulation of already expanded T cells in the absence of appropriate co-stimulation can lead to the efficient induction of activation-induced cell death (AICD) (Takase et al., 2007). The AICD of T lymphocytes is a critical mechanism for eliminating cen- tral and peripheral activated lymphocytes in the immune system and maintaining immune tolerance and homeostasis. (Jiang et al., 2009; Tang et al., 2004). CD4+ T cells from aged animals exhibit a significant age-related decline of function, including weaker proliferative responses, lower IL-2 production, and a reduced ability to help B cells, indicating that the aged-related defects are intrinsic to the decline of CD4+ T cell function (Linton and Dorshkind, 2004; Marko et al., 2007). Recent studies suggest that the decline of aged CD4+ T cell function is associated with autoimmune and infectious disease (Aprahamian et al., 2008; Lages et al., 2008; Prelog, 2006). AICD in T cells is primarily mediated by CD95 and subse- quent CD95 ligand-induced apoptosis (Klemke et al., 2009). The ligation of CD95 by the CD95 ligand results in recruitment of the adaptor molecule Fas-associated protein with death domain (FADD), pro-caspase-8 (also called FLICE), pro-caspase-10, and cel- lular caspase-8 (FLICE)-like inhibitory protein (c-FLIP), formation of the death-inducing signaling complex (DISC) and further cleavage of pro-caspase-8 to active caspase-8. Active caspase-8 then initi- ates the apoptotic program (Bouillet and O’Reilly, 2009; Peter et al., 2005).
In addition to the apoptosis signaling pathway, CD95 ligation also induces the activation of the IKK-NF-nB pathway and the
regulation of the immune system (Wang et al., 2007).The InB kinase (IKK) complex contains two catalytic subunits, IKKα and IKKβ (IKK-1 and IKK-2), and an essential regulatory subunit, IKKγ/NEMO (Bonizzi and Karin, 2004; Israel, 2010). IKK can be activated by a variety of stimuli, including inflammatory cytokines. Once activated by phosphorylation, IKK phosphorylates InBs, a group of cytoplasmic inhibitors of NF-nB, on specific serine residues (Ser32 and Ser36 in InBα and Ser19 and Ser21 in InBβ), and InBs are subsequently ubiquitinated and degraded via the pro- teasomal pathway (Israel, 2010; Perkins, 2007). The freed NF-nB then translocates into the nucleus to regulate the transcription of specific target genes, such as cytokines, chemokines and inhibitors of apoptosis (Bonizzi and Karin, 2004; Israel, 2010). Recent studies show that the IKK pathway can negatively or positively regulate c- Jun N-terminal protein kinase (JNK) activation depending on the nature of the stimulus (Kato et al., 2008; Sakurai et al., 2006; Thomas et al., 2009). JNK is a member of the mitogen-activated protein kinase (MAPK) superfamily and has been suggested to have a critical role in stress-induced apoptosis in a context-dependent manner (Bogoyevitch et al., 2010; Sunayama et al., 2005). JNK has two ubiquitously expressed isoforms, JNK1 and JNK2, and a tissue- specific isoform, JNK3 (Wagner and Nebreda, 2009). Between JNK1 and JNK2, JNK1 is the main c-Jun kinase (Liu et al., 2004; Wagner and Nebreda, 2009).
It is well established that the intricate interplay between the IKK and JNK pathways determines the outcomes of TNF-α signaling. IKK promotes cell survival, whereas prolonged JNK activation enhances cell death (Lin, 2006; Wagner and Nebreda, 2009). IKK induces expression of various inhibitors of apoptosis including c-FLIPL, which specifically inhibits the key initiation steps in caspase-8 activation and thereby blocks TNF-α-induced programmed cell death (Lin, 2006). When IKK/NF-nB activation is abrogated, TNF- α induces prolonged JNK activation, which promotes cell death via several mechanisms, including phosphorylation and activation of the E3 ubiquitin ligase Itch, which specifically ubiquitinates c-FLIP and induces its proteasomal degradation and the apoptosis of the cell (Chang et al., 2006). Meanwhile, prolonged JNK activation is also able to phosphorylate 14-3-3 and release the pro-apoptotic protein Bad from 14-3-3, thereby making cells more susceptible to apoptotic signals (Sunayama et al., 2005). However, the role of IKK inhibition of JNK activation in the age-related apoptosis of T cells is not clear. We report here that CD4+ T cells in aged mice are resistant to AICD induced by anti-CD3 plus IL-2 stimulation. This resistance is likely due to enhanced IKKβ activity in aged CD4+ T cells and the consequent JNK inactivation, which thereby blocks the extrinsic and intrinsic apoptotic pathways. Thus, IKK antago- nizes activation-induced cell death of CD4+ T cells in aged mice via inhibition of JNK activation.
2. Materials and methods
2.1. Reagents
The following antibodies were purchased from Santa Cruz Biotechnology (CA, USA): rabbit polyclonal antibodies against IKKβ (C20), IKKγ (FL419), PARP (B10), IkBα (FL), Bad (C20), Bcl-XL (S18), 14-3-3 (K19) and c-FLIPL (H150). Antibody against JNK was pur- chased from BD Biosciences PharMingen (NJ, USA). Anti-caspase-8 (11G10), c-Jun, phospho-c-Jun (Ser73), and phospho-IkBα (Ser32) antibodies were from cell signaling (MA, USA), and the anti- phospho-14-3-3 (Ser185) antibody was purchased from Thermo Scientific (Rockford, IL, USA). The specific IKK inhibitor PS1145, anti-β-actin antibody and IL-2 were purchased from Sigma (MO, USA). Protein A-sepharose was from GE Healthcare (NJ, USA). RPMI 1640 medium was from Invitrogen Gibco (CA, USA).
2.2. Treatment of mice
Young (2-month-old) and aged (18- to 24-month-old) C57BL/6J mice were purchased from the Laboratory Animal Center, Chinese Academy of Medical Sciences, CAMS. The mice were raised in the Animal Care Facility at the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, in a specific pathogen-free (SPF) environment.
2.3. CD4+ T cell isolation and activation-induced cell death
Spleens from the young and aged mice group (each group including six young or aged mice) were removed, cut into small pieces and processed through a 200-gauge stainless steel mesh. After washing once with RPMI 1640 medium by centrifugation at 300 × g for 10 min, the pellet was re-suspended in RPMI 1640 and carefully laid over Lymphocyte M (Cedarlane Laboratories Limited, Canada). After centrifugation at 800 × g for 30 min, mononuclear cells were collected and washed once with RPMI 1640 medium. Cell viability was more than 95% in all cells when assayed by trypan blue exclusion. A mouse CD4+ T cell enrichment column (R&D Systems, Minneapolis, MN, USA) was employed to separate the splenic CD4+ T cells according to the directions of the manufacturer. Flow cytom- etry was used to analyze cell purity (>95%). Single-cell suspensions (1 × 106 cells) were either unstimulated or stimulated with anti- CD3 (1 µg/ml, Abcam, UK) plus IL-2 (10 ng/ml, Sigma, MO, USA) for the indicated time periods and were cultured at 37 ◦C in 5% CO2 in RPMI 1640 complete medium containing 10% FBS, 100 U/ml peni- cillin, 50 µg/ml streptomycin, 2 mM glutamine, and 20 mM HEPES.
2.4. Measurements of cell apoptosis
The apoptosis of young and aged CD4+ T cells was measured by flow cytometry. Briefly, cells were collected at indicated time points, washed with phosphate buffered saline (PBS), and re- suspended in binding buffer (50 mM Tris, 100 mM NaCl, 1% BSA, 0.02% sodium azide, pH 7.4). The cell concentration was adjusted to 5 × 105/ml. A volume of 5 µl of annexin V was added into 195 µl of cell suspension, and the mixture was placed at room tempera- ture for 10 min. After washing, cells were re-suspended in 190 µl binding buffer, and 10 µl of PI (20 µg/ml) was added into the sus- pension. Apoptosis was analyzed by flow cytometry (FACS Calibur, Becton-Dickinson, USA) with annexinV−/PI+, annexinV+/PI−, and annexinV+/PI+ representing dead cells, early stage apoptotic cells, and late stage apoptotic cells, respectively.
2.5. Analysis of DNA fragmentation
Cells were harvested, and DNA was prepared as previously described (Yuan et al., 2008). Briefly, 1 × 107 cells were washed in PBS and re-suspended in 50 µl of 50 mM Tris–HCl pH 8.0, 10 mM EDTA and 0.5 mg/ml proteinase K (Merck, Darmstadt, Germany). After incubation at 50 ◦C for 1 h, 1 µl of 10 mg/ml RNaseA (Invitro- gen, CA, USA) was added for an additional 1 h at 37 ◦C. The samples were mixed with 25 µl of 10 mM EDTA (pH 8.0) containing 1% (w/v) low melting point agarose, 0.25% bromophenol blue and 40% sucrose at 70 ◦C. The DNA was separated in 2% agarose gels and visualized by UV illumination after ethidium bromide staining.
2.6. Cytokine assays
Young and aged CD4+ T cells were plated at 2 × 106 cells/well in 96-well plates. Cells were stimulated with anti-CD3 (1 µg/ml, Abcam, UK) plus IL-2 (10 ng/ml, Sigma, MO, USA) for 36 h. The cul- ture supernatants were collected, and the levels of IL-6 and TNF-α in the culture medium were measured using commercially avail- able ELISA assay kits (BD Biosciences mouse IL-6 or TNF-α ELISA kit, USA) according to the manufacturer’s protocol.
2.7. Immunoblot assay
Immunoblotting was performed as described previously (Deng et al., 2008). Briefly, cells were washed with PBS and were lysed in lysis buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 10 mM β-glycerophosphate, 5 mM EGTA, 1 mM sodium pyrophosphate, 5 mM NaF, 1 mM Na3VO4, 0.5% Triton X-100, and 1 mM DTT) supplemented with protease inhibitors (1 mM PMSF, 5 µg/ml leu- peptin, 5 µg/ml pepstatin A, and 5 µg/ml aprotinin). Proteins were separated by SDS-PAGE and were electrically transferred to a polyvinylidene difluoride membrane. The membranes were blocked for 1 h at room temperature in TBS (150 mM NaCl, 20 mM Tris, pH 7.5, 0.05% Tween-20) containing 5% milk and probed with specific first antibodies for 1 h at room temperature. Blots were then incubated with HRP-conjugated anti-rabbit (7074, Cell Sig- naling) or anti-mouse (7076, Cell Signaling) secondary antibody. Immunoreactive proteins were visualized using the ECL detection system (Amersham, USA).
Fig. 1. Resistance of aged CD4+ T cells to activation-induced cell death (AICD). (A) Splenic CD4+ T cells were separated from young and aged C57BL/6J mice and stained with annexin V/PI after stimulation with anti-CD3 (1 µg/ml) plus IL-2 (10 µg/ml) for the time indicated. The apoptotic rate was determined by flow cytometry. The results are presented as mean ± standard error and represent three individual experiments. (* p < 0.01 compared to young 0 h; # p < 0.01 compared to aged 0 h). (B) The genomic DNA was extracted from young and aged CD4+ T cells after AICD for 24 h, and DNA fragmentation was analyzed by agarose gel electrophoresis followed by staining with ethidium bromide. (C) AICD was induced by anti-CD3/IL-2 in young and aged CD4+ T cells for 16 or 32 h, and caspase-3 activity was determined as described in the materials and methods (* p < 0.01 compared to young 0 h; # p < 0.01 compared to aged 0 h; Δ, p < 0.05 compared to aged 0 h). (D) AICD was induced in young and aged CD4+ T cells for the time indicated, and the cleavage of PARP and the expression level of β-actin were detected by immunoblotting using anti-PARP or anti-β-actin antibodies. 2.8. Protein kinase assay Immune complex kinase assays and glutathione S-transferase (GST)-IkBα and GST-c-Jun pull down kinase assays were performed as described previously (Herr et al., 2000). Briefly, the lysates of CD4+ T cells were used for immunoprecipitation with IKKγ or JNK antibodies and protein A-sepharose beads. Kinase assays were carried out in the presence of 20 mM HEPES (pH 7.6), 20 mM MgCl2, 1 mM DTT, 17 µM non-radioactive ATP, and GST-IkBα or GST-c-Jun (2–4 µg) for 60 min at 30 ◦C. Reactions were terminated by the addition of 4 × SDS sample buffer and heating at 95 ◦C for 5 min. The proteins were separated by 12% SDS-PAGE, and phosphorylation of GST-IkBα and GST-c-Jun were detected by immunoblotting using antibodies against phospho-IkBα (Ser32) and phospho-c-Jun (Ser73). 2.9. Coimmunoprecipitation Young and aged CD4+ T cell lysates (400 µg of protein) were incubated with 40 µl (1:1 slurry) of protein A-sepharose and 4 µg of polyclonal anti-14-3-3 or anti-Bad antibody overnight at 4 ◦C. As a control, protein A-sepharose only was incubated with antibodies overnight at 4 ◦C. Following incubation, beads were washed twice with lysis buffer. Proteins were eluted with SDS sample dilution buffer and separated by 12% SDS-PAGE. Immunoprecipitated Bad and 14-3-3 were detected by immunoblotting. 2.10. Caspase activity assay CD4+ T cells (1 × 107 per milliliter) were treated with anti-CD3 plus IL-2 stimulation for the indicated time, washed three times with PBS, and lysed in a buffer containing 10 mM Tris, pH 7.5, 130 mM NaCl, 1% Triton X-100, 10 mM NaPi, and 10 mM NaPPi. Cell lysates were centrifuged at 13,000 × g, and the cleared super- natants were collected for protease assays. The lysates (50 µg) and 10 µg Ac-DEVD-AFC (BD Biosciences, NJ, USA) were added to 1 ml protease assay buffer and incubated for 1 or 2 h at 37 ◦C. Caspase- 3 activity was determined by monitoring the release of AFC at an excitation of 400 nm and an emission wavelength of 505 nm using a cytofluorometer. 2.11. Statistical analysis All statistical analyses were performed using Student’s t-test. A probability level of 0.05 or less was considered to be significant. Data are presented as mean value ± standard error. 3. Results 3.1. Resistance of aged CD4+ T cells to activation-induced cell death (AICD) Recent studies suggest that the age-related progressive accu- mulation of cellular damage leads to a decrease in apoptosis in mammalian tissues (Warner, 2007; Zhang and Herman, 2002). To further confirm the age-related decline in apoptosis in mice, we separated splenic CD4+ T cells from young and aged mice for induc- tion of AICD and determined the capacity of the activated CD4+ T cells to undergo apoptosis. An apoptotic cell death assay revealed that the AICD response of aged CD4+ T cells was significantly lower than that of young cells after stimulation with anti-CD3 and IL-2 (Fig. 1A). Thirty-two hours after induction of AICD, the apoptotic rate of young CD4+ T cells was about 85% while that of aged cells was only 51%. A DNA fragmentation assay also showed that the genomic DNA extracted from aged CD4+ T cells underwent much less degradation than DNA extracted from young cells (Fig. 1B). Furthermore, a caspase-3 assay revealed that there was markedly reduced caspase-3 activity in aged CD4+ T cells after AICD (Fig. 1C), which is consistent with the decline of apoptosis in aged CD4+ T cells (Fig. 1A). Activation of caspase-3 results in the cleavage of a number of important cellular proteins, including poly-ADP- ribose polymerase (PARP). Immunoblotting results demonstrated that there was decreased cleavage of PARP in aged CD4+ T cells in response to anti-CD3 plus IL-2 stimulation compared to young CD4+ T cells (Fig. 1D). Taken together, these data suggest that the aged CD4+ T cells are resistant to activation-induced cell death. Fig. 2. Inflammation-induced IKKβ activation antagonizes AICD of aged CD4+ T cells. (A and B) Young and aged splenic CD4+ T cells were unstimulated or stimulated with anti-CD3 (1 µg/ml) plus IL-2 (10 µg/ml) for 18 and 36 h, and the cytokines IL-6 (A) and TNF-α (B) were measured in the culture supernatants by ELISA (* p < 0.01 compared to aged 0 h). (C) Young and aged splenic CD4+ T cells were stimulated with anti-CD3 plus IL-2 for 12 h, and the IKKβ activity was measured by immune complex kinase assay using GST-InB as a substrate. The phosphorylation of GST-InB was monitored by immunoblotting using an anti-phospho-InB-Ser32 antibody. The expression levels of IKKβ and InBα were detected by immunoblotting using anti-IKKβ or InB antibodies, respectively. (D) Aged CD4+ T cells were pretreated with the specific IKK inhibitor PS1145 at various doses for 45 min followed by stimulation with anti-CD3 plus IL-2 for 12 h. The activity and the expression level of IKKβ were measured as described in (C). (E) Young and aged splenic CD4+ T cells were pretreated without or with the IKK inhibitor PS1145 followed by stimulation with anti-CD3 plus IL-2 for 0, 16 or 32 h, and the apoptotic rate was determined as described in Fig. 1A (# p < 0.01 compared to PS1145 untreated aged 16 h; * p < 0.01 compared to PS1145 untreated aged 32 h). 3.2. Inflammation-induced IKKˇ activation antagonizes AICD of aged CD4+ T cells Recent studies have indicated that during aging, there is an age- related pro-inflammatory phenotype (Franceschi et al., 2007; Fulop et al., 2006) and that the inflammation signals are mostly mediated through the NF-nB system via IKKβ (Bonizzi and Karin, 2004; Tang et al., 2001). To test whether aged mouse CD4+ T cells are associated with an age-related pro-inflammatory phenotype, CD4+ T cells sep- arated from young and aged spleens were stimulated with anti-CD3 plus IL-2, and the cytokines secreted into the media were analyzed. As expected, there were significantly increased pro-inflammatory cytokine levels, namely, IL-6 and TNF-α, in the anti-CD3 plus IL- 2 stimulated culture medium of aged CD4+ T cells compared to young CD4+ T cells at 18 and 36 h (Fig. 2A and B), which is consis- tent with previous reports (Franceschi et al., 2007; Johnson, 2006). Meanwhile, an immune complex kinase assay indicated that IKKβ activity was enhanced after AICD in aged CD4+ T cells compared to young cells, and even the basal IKKβ activity was elevated in aged CD4+ T cells (Fig. 2C, upper panel). We next asked whether the difference in IKKβ kinase activity between young and aged CD4+ T cells resulted from variation in the expression level of IKKβ. Interestingly, after AICD, the expres- sion level of IKKβ in aged CD4+ T cells was significantly higher than that in young cells, as demonstrated by immunoblotting using an anti-IKKβ antibody (Fig. 2C, lower panel). To further inves- tigate the role of IKKβ in the AICD of CD4+ T cells, a specific IKK inhibitor, PS1145, was used to inhibit IKKβ activity in both young and aged CD4+ T cells, and AICD was analyzed. The in vitro immune complex kinase assay revealed that pretreatment of aged CD4+ T cells with PS1145 inhibited anti-CD3/IL-2-induced IKKβ activation in a dose-dependent manner (Fig. 2D). A concentration of 10 µM of PS1145 significantly inhibited the IKK activity and resulted in a dramatic increase of anti-CD3/IL-2-induced AICD in aged CD4+ T cells (Fig. 2E). Taken together, the above data suggest that inflammation-induced IKKβ activation antagonizes AICD of aged CD4+ T cells. Fig. 3. JNK regulates IKK-mediated antagonism of AICD in aged CD4+ T cells through the extrinsic and intrinsic apoptotic pathways. Young and aged splenic CD4+ T cells were stimulated without or with anti-CD3 plus IL-2 for 12 h. (A) The JNK activity was measured by immune complex kinase assays using GST-c-Jun as a substrate, and the phosphorylation of GST-c-Jun was monitored by immunoblotting using an anti-phospho-c-Jun-Ser73 antibody. The expression level of JNK and c-Jun were detected by immunoblotting using anti-JNK or anti-c-Jun antibodies, respectively. (B) c-FLIPL expression level and cleaved caspase-8 in young and aged splenic CD4+ T cells were monitored by immunoblotting using anti-c-FLIPL or anti-caspase-8 antibodies, respectively. (C) The phosphorylation and expression level of 14-3-3-β in young and aged splenic CD4+ T cells were measured by immunoblotting using anti-phospho-14-3-3-β-Ser185 or anti-14-3-3-β antibodies, respectively. (D) Cell extracts of young and aged splenic CD4+ T cells were subjected to immunoprecipitation with anti-14-3-3-β antibody, and the amount of Bad-associated with 14-3-3 was determined by immunoblotting with an anti-Bad antibody. Total 14-3-3-β proteins in the cell extracts were analyzed by immunoblotting with an anti-14-3-3-β antibody. (E) Cell extracts of young and aged CD4+ T cells were subjected to immunoprecipitation with anti-Bad antibody, and the amount of Bcl-XL associated with Bad was determined by immunoblotting with an anti-Bcl-XL antibody. Total Bad proteins in the cell extracts were analyzed by immunoblotting with an anti-Bad antibody. 3.3. JNK regulates IKK-mediated antagonism of AICD in aged CD4+ T cells through extrinsic and intrinsic apoptotic pathways To investigate the mechanism by which IKKβ antagonizes AICD of aged CD4+ T cells, we sought to determine the role of JNK in the AICD of aged CD4+ T cells because JNK is a well-known kinase that mediates apoptotic signals and because IKKβ-mediated NF- nB activation is able to inhibit the function of JNK (Lin and Karin, 2003; Perkins, 2007). An immune complex kinase assay indicated that JNK activity was significantly decreased in aged CD4+ T cells after AICD (Fig. 3A, upper panel), this was not the result of varia- tion in JNK expression, as demonstrated by immunoblotting with an anti-JNK antibody (Fig. 3A, lower panel). c-FLIPL is homologous to caspase-8 except that it lacks caspase activity, and thus, it can block the extrinsic apoptotic pathway by preventing caspase-8 activation (Chang et al., 2006). Immunoblotting results showed that there was no visible change in c-FLIPL expression or caspase-8 activation in aged CD4+ T cells after AICD. In contrast, the expression level of c- FLIPL was greatly decreased, and caspase-8 was activated in young CD4+ T cells after AICD (Fig. 3B). Thus, IKK-mediated JNK inactiva- tion results in prolonged c-FLIPL expression and inhibition of the extrinsic apoptotic pathway in aged CD4+ T cells. Because JNK-mediated phosphorylation of 14-3-3 promotes the association of Bad with Bcl-XL and induces cell apoptosis through the intrinsic apoptotic pathway (Sunayama et al., 2005), we next asked whether JNK phosphorylation of 14-3-3 affects the inter- action between 14-3-3 and Bad as well as the association of Bad and Bcl-XL in both young and aged CD4+ T cells. Immunoblotting results showed that the elevated JNK activity in response to AICD in young CD4+ T cells dramatically increased phosphorylation of 14-3-3-β at Ser185. However, the 14-3-3-β-Ser185 phosphorylation was abolished in aged CD4+ T cells (Fig. 3C). A coimmunoprecipitation assay revealed that when endogenous 14-3-3-β was immunopre- cipitated from lysates of unstimulated young and aged CD4+ T cells, endogenous Bad was coprecipitated (Fig. 3D). However, the amount of Bad that coprecipitated with 14-3-3-β was markedly reduced in young CD4+ T cells in response to AICD. Under the same conditions, there was no detectable change in the 14-3-3-β and Bad interaction in aged CD4+ T cells (Fig. 3D). Furthermore, the association of Bad and Bcl-XL was only detected in young CD4+ T cells after anti-CD3 plus IL-2 stimulation (Fig. 3E), which corresponds to the reduction of 14-3-3-β association with Bad (Fig. 3D) and the increased apop- totic rate in young CD4+ T cells (Fig. 1A). Altogether, these data suggest that JNK regulates IKK-antagonized AICD of aged CD4+ T cells through the extrinsic and intrinsic apoptotic pathways. 3.4. Blocking of IKK activity sensitizes the aged CD4+ T cells to AICD The fact that IKK has a key role in the resistance of aged CD4+ T cells to AICD prompted us to investigate whether blocking IKK activity could reverse the AICD-resistant phenotype in aged CD4+ T cells. Indeed, pretreatment of aged CD4+ T cells with 10 µM of the IKK inhibitor PS1145 resulted in a significant increase of AICD-induced JNK activity (Fig. 4A, lower panel). Under the same conditions, PS1145 treatment induced a degradation of the anti- apoptotic protein c-FLIPL and the activation of caspase-8 in aged CD4+ T cells (Fig. 4B), which is consistent with the AICD result in aged CD4+ T cells treated with PS1145 (Fig. 2E). Furthermore, the phosphorylation of 14-3-3-β at Ser185 was increased in aged CD4+ T cells after IKK activity was inhibited by PS1145 (Fig. 4C). The amount of Bad that coprecipitated with 14-3-3-β from lysates of PS1145-treated aged CD4+ T cells was decreased (Fig. 4D, upper panel). However, the amount of Bcl-XL that coprecipitated with Bad from the lysates of PS1145-treated aged CD4+ T cells was increased (Fig. 4D, lower panel), which corresponded to the reduction in the association of 14-3-3-β with Bad. PS1145 treatment also signifi- cantly increased caspase-3 activity in aged CD4+ T cells (Fig. 4E), thus sensitizing the aged T cells to AICD. Altogether, the above data suggest that blocking the IKK activity by PS1145 sensitizes the aged CD4+ T cells to AICD. Fig. 4. Blocking IKK activity sensitized the aged CD4+ T cells to AICD. Aged splenic CD4+ T cells were pretreated without or with the specific IKK inhibitor PS1145 (10 µM) for 45 min followed by stimulation with anti-CD3 plus IL-2 for 12 h. (A) The activity and expression levels of IKKβ and JNK were detected as described in Figs. 2C and 3A, respectively. (B) c-FLIPL expression levels and cleaved caspase-8 were measured by immunoblotting using anti-c-FLIPL or anti-caspase-8 antibodies, respectively. (C) The phosphorylation and expression level of 14-3-3-β were measured by immunoblotting as described in Fig. 3C. (D) 14-3-3-β-associated Bad and Bad-associated Bcl-XL were analyzed as described in Fig. 3C and D. (E) Aged splenic CD4+ T cells were pretreated without or with the specific IKK inhibitor PS1145 (10 µM) for 45 min followed by stimulation with anti-CD3 plus IL-2 for 16 or 32 h. Caspase-3 activity was measured as described in Fig. 1C (* p < 0.01 compared to PS1145 untreated 16 h; # p < 0.01 compared to PS1145 untreated 32 h). 4. Discussion Apoptosis following stimulation and proliferation has a critical role in the regulation of T cell responses to stimuli and preventing accumulation of dysfunctional senescent T cells during aging (Fulop et al., 2006). However, the apoptosis of T cells during aging in the murine system is controversial. T cells from C57BL/6 mice have shown increased apoptosis upon anti-CD3/anti-CD28 stimulation (Han et al., 2006). In contrast, splenic T cells from C57BL/6 aged mice have shown decreased apoptosis (Hsu et al., 2001). Other reports have also indicated that during aging, a decline in T cell apopto- sis leads to the accumulation of damage and functional defects (Pawelec et al., 2001; Zhang and Herman, 2002). In this report, we demonstrate that the AICD of aged mouse CD4+ T cells is attenu- ated (Fig. 1), likely due to enhanced IKKβ activity in aged CD4+ T cells and the consequent JNK inactivation, which thereby blocking the extrinsic and intrinsic apoptotic pathways. Thus, the defect in the AICD of aged CD4+ T cells contributes largely to the age-related decline of T cell function. During aging, an imbalance between adaptive and innate immu- nity leads to a pro-inflammatory phenotype called inflamm-aging (Franceschi et al., 2007). There is a body of evidence on the age- related pro-inflammatory phenotype (Franceschi et al., 2007; Fulop et al., 2006). Our finding also demonstrated that pro-inflammatory cytokines, such as IL-6 and TNF-α, were dramatically increased in aged mice (Fig. 2A and B), consistent with the previous report (Johnson, 2006). TNF-α is a strong inducer of IKKβ activation, and the inflammatory signals are primarily mediated through the NF- nB system via IKKβ (Israel, 2010). Our results indicated that IKKβ activity in aged CD4+ T cells was enhanced after AICD (Fig. 2C), which may result from an increased expression level of IKKβ and increased TNF-α secretion (Fig. 2B and C). In addition, reduced caspase-3 activity could also contribute to the enhanced IKKβ activity in aged CD4+ T cells (Fig. 1C) because IKKβ is specifically proteolyzed by caspase-3 and promotes TNF-α-induced cell death (Tang et al., 2001). Thus, increased IKKβ activity contributed to the decline of AICD in aged CD4+ T cells. Moreover, blocking IKK activa- tion by PS1145 induced a significant increase in AICD in aged CD4+ T cells (Fig. 2C and E), suggesting that the defect in AICD in aged CD4+ T cells may result in an accumulation of reactive T cells, and the ineffective elimination of self-reactive T cells ultimately leads to inflammation in aged mice. The IKK-NF-nB pathway induces expression of various inhibitors of apoptosis, including c-FLIPL, which specifically inhibits the key initiation steps of caspase-8 activation and thereby blocks CD95-induced AICD of T cells (Jiang et al., 2009). Previous data have demonstrated JNK-mediated phosphorylation and activation of the E3 ubiquitin ligase Itch, which specifically ubiquitinates c-FLIP and induces its proteasomal degradation (Chang et al., 2006). Our data demonstrated that there was no obvious change in c-FLIPL expres- sion in aged CD4+ T cells after AICD, but the expression level of c-FLIPL in young CD4+ T cells was significantly decreased (Fig. 3B). Under the same conditions, there was increased IKKβ activity in aged CD4+ T cells associated with abolished JNK activation. These results indicated that elevated IKKβ and c-FLIPL expression might be a key factor in inhibiting aged CD4+ T cell apoptosis. It has been demonstrated that IKKβ and c-FLIPL promote inflammation and cell proliferation as well as inhibiting apoptosis (Krueger et al., 2001; Perkins, 2007). Thus, elevated IKKβ and c-FLIPL expression may have an important role in promoting the resistance to AICD and the inflammatory status in aged mice. Accumulating evidence has shown that JNK has a critical role in both cell survival and apoptosis. In the absence of NF-nB activation, JNK phosphorylation of 14-3-3 and release of the pro-apoptotic protein Bad from 14-3-3 induces apoptosis (Sunayama et al., 2005). However, there is evidence that JNK also contributes to cell survival. Recent studies have shown that JNK directly phosphorylates Bad at Thr201 to reduce the affinity of Bad for Bcl-2/Bcl-XL and inactivate Bad (Deng et al., 2008; Yu et al., 2004). We found that after AICD, IKK-mediated JNK inactivation reduced the interaction of Bad with Bcl-XL but increased the affinity of Bad for 14-3-3 in aged CD4+ T cells, thus resulting in a resistance to AICD in aged CD4+ T cells. This finding suggests that JNK may function as an apoptosis medi- ator rather than a survival factor for CD4+ T cells during aging. A recent study demonstrated that naïve CD4+ T cells from aged mice are intrinsically longer-lived and that this longer lifespan of aged cells was independent of self-peptide–MHC interaction and leads to the development of age-associated defects (Tsukamoto et al., 2009). Further studies are needed to examine whether IKK-mediated JNK inactivation has any role in the age-associated increase in the lifes- pan of naïve CD4+ T cells. 5. Conclusions Here we show that CD4+ T cells in aged mice display increased IKKβ activity that is associated with attenuated JNK activation. The IKKβ-mediated JNK inactivation in aged CD4+ T cells reduces the degradation of c-FLIPL and the association of Bad with Bcl-XL, but it increases the affinity of Bad for 14-3-3. Blocking IKKβ activity sensitizes the aged CD4+ T cells to AICD. Thus, IKK antagonizes the AICD of CD4+ T cells in aged mice via PS-1145 inhibition of JNK activation.