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Originally published In Press as doi:10.1074/jbc.M606721200 on January 9, 2007

J. Biol. Chem., Vol. 282, Issue 10, 7416-7423, March 9, 2007
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Essential Role for Caspase-8 in Toll-like Receptors and NF{kappa}B Signaling*Formula

Bénédicte Lemmers{ddagger}§, Leonardo Salmena{ddagger}§, Nicolas Bidère||, Helen Su||, Elzbieta Matysiak-Zablocki{ddagger}§, Kiichi Murakami{ddagger}§, Pamela S. Ohashi{ddagger}§, Andrea Jurisicova**, Michael Lenardo||, Razqallah Hakem, Supported by a salary award from the Canadian Institute of Health Research{ddagger}§1, and Anne Hakem{ddagger}§2

From the {ddagger}Ontario Cancer Institute, §The Advanced Medical Discovery Institute, and the Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2C1, Canada, the ||Laboratory of Immunology, NIAID, National Institutes of Health, Bethesda, Maryland 20892, and the **Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 2X1, Canada

Received for publication, July 14, 2006 , and in revised form, November 29, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In addition to its pro-apoptotic function in the death receptor pathway, roles for caspase-8 in mediating T-cell proliferation, maintaining lymphocyte homeostasis, and suppressing immunodeficiency have become evident. Humans with a germline point mutation of CASPASE-8 have multiple defects in T cells, B cells, and NK cells, most notably attenuated activation and immunodeficiency. By generating mice with B-cell-specific inactivation of caspase-8 (bcasp8-/-), we show that caspase-8 is dispensable for B-cell development, but its loss in B cells results in attenuated antibody production upon in vivo viral infection. We also report an important role for caspase-8 in maintaining B-cell survival following stimulation of the Toll-like receptor (TLR)2, -3, and -4. In response to TLR4 stimulation, caspase-8 is recruited to a complex containing IKK{alpha}beta, and its loss resulted in delayed NF{kappa}B nuclear translocation and impaired NF{kappa}B transcriptional activity. Our study supports dual roles for caspase-8 in apoptotic and nonapoptotic functions and demonstrates its requirement for TLR signaling and in the regulation of NF{kappa}B function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Caspase-8 has been best characterized as a cysteine protease that cleaves specific substrates to transmit apoptotic signals downstream of death receptors (DR)3 (1–4). During DR signaling, procaspase-8 is recruited to the death-inducing signaling complex (DISC), where oligomerization drives its own activation by self-cleavage to form an activated caspase-8 tetramer complex in a process termed "proximity-induced activation" (5). Once activated, caspase-8 mediates apoptosis through a cell-specific type I or type II DR pathway. In type I cells, active caspase-8 at the DISC can directly process and activate caspase-3, leading to apoptosis. In type II cells, caspase-8 processes the BH3-only molecule Bid to form a truncated Bid molecule that translocates to the mitochondrial membrane and triggers cell death through the mitochondria (6).

In addition to their apoptotic roles, a new paradigm for caspases in non-apoptotic roles has become clear. In various studies, activation of caspases was detected in non-apoptotic-activated lymphocytes (7–10). Additionally, lymphocyte activation and proliferation was defective in the presence of selective caspases inhibitors (11–14). T-cell-specific deletion or overexpression of a dominant negative form of Fas-associated death domain protein (FADD), another DISC component, demonstrated its requirement for proper T-cell activation and IL2-dependent proliferation (15–18). Together these findings implicate caspases and other DISC components in non-apoptotic functions.

Until recently, embryonic lethality prevented the generation and phenotypic characterization of adult mice missing caspase-8, necessitating tissue-specific inactivation (19–21). We recently reported the conditional inactivation of caspase-8 in the T-cell lineage (tcasp8-/-) and identified important novel non-apoptotic roles for caspase-8 in maintaining peripheral T-cell homeostasis, in mediating expansion of T-cells upon in vitro activation, and in inducing an effective in vivo T-cell immune response against viral infections (20). Studies of human patients with a caspase-8 point mutation also support a non-apoptotic role for caspase-8. These patients are immunodeficient, show reduced activation of B, T, and NK cells in vitro, exhibit decreased levels of circulating antibodies and impaired immunoglobulin production in response to antigenic stimulation (22). Impaired activation of T, B, and NK cells from these patients was associated with defective NF{kappa}B nuclear translocation (23). In response to TCR stimulation of T cells, caspase-8 contributes to the formation of the activation receptor-induced signalosome (ARIS) that contains CARMA1-BCL10-MALT1 and the I{kappa}B kinase (IKK) complexes. Loss of caspase-8 impairs ARIS formation leading to defective NF{kappa}B signaling in response to TCR stimulation. Toll-like receptors are important components of innate immunity and provide a first-line of defense against microbial infections. Binding of specific TLRs by their appropriate antigens activates B cells, induces their antigen-presenting ability, and stimulates the production of neutralizing antibodies (24, 25).

At least 10 members of the Toll-like receptor (TLR1–10) family have been identified in human, and 13 in mice (26). Examples of such antigens activating specific TLRs are: bacterial lipoprotein for TLR2, double-stranded viral RNA (or analog, poly(I:C)) for TLR3, bacterial lipopolysaccharide (LPS) for TLR4, flagellin for TLR5, single-stranded DNA for TLR7, and unmethylated CpG DNA for TLR9 (13, 27, 28).

A link between TLR pathways and DISC components has been identified in recent studies of caspase-8, FADD, Flip (FLICE-like inhibitory protein), and RIP-1 (receptor-interacting protein-1) (29–32). Flip is recruited to the DISC and functions as an endogenous dominant-negative molecule by inhibiting FADD-caspase-8 interactions and thereby inhibits DR apoptosis (33). Deletion of FADD or FLIP in mouse embryonic fibroblats (MEFs) enhanced NF{kappa}B activation in response to LPS stimulation (29, 30), whereas RIP-1 deficiency resulted in decreased NF{kappa}B activation or survival following TLR3 or TLR4 stimulation, respectively (31, 32). The molecular mechanisms linking FADD, Flip, and RIP-1 with TLR pathways remain to be identified.

In this study, we have investigated the roles of caspase-8 in development, apoptosis, and proliferation of B lymphocytes. As expected, caspase-8-deficient B cells were resistant to CD95-mediated killing; however unlike T lymphocytes (20), we found that caspase-8 is not required for development and homeostasis of B lymphocytes. Our study identifies an impaired response of caspase-8-deficient B cells to TLR4 and links this defect to a delayed nuclear translocation of NF{kappa}B, and defective transcription of its target genes in the absence of caspase-8. We also report that loss of caspase-8 leads to reduced phosphorylation of p65 at serine 536, which is known to regulate p65 nuclear translocation (34). Finally, in response to TLR4 stimulation, caspase-8 was found to be transiently recruited to a complex that contains IKK{alpha}beta thereby placing this caspase in a signaling complex important for cell survival and immune responses.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Mice—Casp8-conditional mutant mice (20) were intercrossed with CD19Cre transgenic mice (35) to obtain bcasp8 mice. All mice studied were in a mixed 129/J x C57BL/6 genetic background and were genotyped by PCR (primer sequences and PCR conditions available upon request). Controls used in the different experiments correspond to either wild-type, casp8fl/wt CD19-Cre, or CD19-Cre mice. The former two genotypes produced similar results compared with the wild-type controls. Mice used for each experiment were littermates. All experiments were performed in compliance within the Ontario Cancer Institute Animal Care Committee Guidelines.

Flow Cytometry Analysis—Single cell suspensions prepared from thymus, spleen, lymph lodes (cervical, inguinal, axillary, brachial, and inguinal lymph nodes were pooled), and bone marrow from mice 8–12 weeks of age were stained with antibody at 4 °C in phosphate-buffered saline, 10% fetal calf serum (Invitrogen). The following antibodies were used to analyze cell populations: anti-CD4, anti-CD8, anti-Thy.1, anti-B220, anti-CD43, anti-IgM, anti-IgD, anti-CD21/35, and anti-CD23. Lymphocytes were analyzed by flow cytometry (FACScalibur; Becton Dickinson) with CellQuest software (Applied Biosystems). Cells were sorted using a FACSVantage flow cytometer (Becton Dickinson).

B-cell Purification and Thymidine Incorporation—For all the experiments, B cells were purified by negative selection or selected by activated cell sorting (FACS). For negative selection, splenocytes were subjected to red blood cell lysis, depleted of macrophages using anti-rat IgG magnetic beads (Dynabeads, Dynal) following anti-CD11b and anti-Gr-1 (eBioscience) labeling. T cells were eliminated using anti-Thy 1.2-bound magnetic beads (Dynabeads, Dynal). All B-cell experiments were performed with purified B cells with a purity ≥90%, LPS (10, 1 and 0.1 µg/ml) (serotype 0111:B4; 055:B5; Sigma), CPG (1 µg/ml) (Coley Pharmaceutical ODN 1826/2138), and poly(IC) (100 µg/ml) (invivoGen). Cells were harvested at 24 or 48 h after an 18-h pulse with [3H]thymidine (1 µCi/well) (Amersham Biosciences), and incorporation of [3H]thymidine was measured with a Matrix 96 direct beta-counter system (Canberra Packard). Lipoteichoic acid (LTA) and ultrapure LPS (invivoGen) were used at 10 µg/ml and 5 µg/ml, respectively.

Cell Cycle and CFSE Analysis—For cell cycle analysis, purified B cells were fixed in 70% ethanol and stained with 5 µg/ml of propidium iodide as previously described (20). Purified splenic B cells (106) were collected in serum-free medium and incubated with CFSE (Molecular Probes-5 µM) at 37 °C for 10 min. Following three washes in Iscove's medium, 10% fetal calf serum, the CFSE-labeled cells were stimulated for 24, 48, and 72 h with LPS (10 µg/ml). For each time point, cells were stained for survival using 7-amino-actinomycin D (7-ADD), and the levels of CFSE incorporation determined by FACS analysis.

Apoptosis—Purified B cells were stimulated with anti-IgM +IL4 (20 µg/ml, 5 ng/ml) for 3 days and then with CD95L (recombinant hCD8-mCD95L fusion protein 1 µg/ml) with or without cycloheximide (0.1 µg/ml) for 24 h. Apoptosis and cell death were measured after treatment using Annexin-V-FITC/PI Apoptosis Detection kit (R&D Systems) or 7-AAD (Sigma).

Vesicular Stomatitis Virus Infections—Mice were immunized with VSV-Indiana (2 x 106 PFU, intravenously). After 4, 8, and 12 days post-infection, sera were collected, and neutralizing IgM and IgG immunoglobulin titers determined as described (36).

Immunofluorescence—Cells (105) were used for cytospin centrifugation. Cytospin specimens of B cells were fixed with 4% PFA for 30 min at room temperature. Cells were incubated with 1:50 dilution of anti-p65 Ab (C-20, Santa Cruz Biotechnology) for 45 min at room temperature. Labeling was revealed using anti-rabbit-conjugated to fluorescein isothiocyanate (Jackson Immunoresearch). Cells were stained with Hoescht for 5 min and then mounted with Fluoromount (Southern Biotec). Images were collected on a Leica TCS-NT/SP confocal microscope (Leica Microsystems) using a x63 oil immersion objective NA 1.32, zoom X.

Immunoprecipitation and Western Blot Analysis—Total protein extracts from purified B cells were prepared as previously described (20). Immunoprecipitation was performed using protein A-Sepharose following incubation of cell extracts with anti-IKK{alpha}beta (H-470, Santa Cruz Biotechnology) and anti-caspase-8 (epitope SNKDDRRNKGKQMP; amino acids 453–466 of murine caspase-8) antibodies. Optimal Ab concentrations and conditions for immunoprecipitation were determined in pilot studies. The separation of cytosol and nucleus was performed as previously described (23). Proteins were separated on 4–20% Tris-glycine gels (Novex). The following antibodies were used for Western blotting in 5% powdered milk (Carnation) in PBS-T: rabbit polyclonal anti-caspase-8 (epitope SNKDDRRNKGKQMP; amino acids 453–466 of murine caspase-8), rat monoclonal anti-caspase-8 antibody (kindly provided by Dr. A Strasser), anti-phospho-p38 MAPK, anti-phospho-IKB{alpha}, anti-IKB{alpha}, anti-PARP, anti-phospho-p65 (S536), and anti-beta-actin (Sigma), and anti-NFkB-p65 (Santa Cruz Biotechnology, C-20).

Real Time PCR—Cell-sorted B cells were stimulated with LPS (10 µg/ml) for 0, 2, 4, and 7 h. Cells were harvested, and total RNA was made using the TRIzol reagent (Invitrogen). cDNA was made using the Invitrogen kit. The SYBR green kit from Applied Biosystems was used for real time PCR. The following oligonucleotides were used for real time PCR: GADPH (5'-AACAGGAAGCCCATCACCATCTT-3' and 5'-GCCCTTCCACAATGCCAAAGTT-3'); TNF-{alpha} (5'-CCCTCACACTCAGATCATCTTCT-3' and 5'-GCTACGACGTGGGCTACAG-3'); IL6 (5'-TAGTCCTTCCTACCCCAATTTCC-3' and 5'-TTGGTCCTTAGCCACTCCTTC-3'), INF-beta (5'-CAGCTCCAAGAAAGGACGAAC-3'and 5'-GGCAGTGTAACTCTTCTGCAT-3') and IP10 (5'-CCAAGTGCTGCCGTCATTTTC-3'and 5'-GGCTCGCAGGGATGATTTCAA-3'). All PCR was performed on the Applied Biosystems 7900HT Fast Real Time PCR System.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To study the function of caspase-8 in B lymphocytes, mice carrying the caspase-8 mutation targeted to the B-cell lineage (casp8fl/fl;CD19Cre, also called bcasp8-/-) were generated by crossing mice homozygous for the caspase-8-floxed allele (casp8fl/fl) (20) with mice expressing the Cre recombinase under the control of the B-cell-specific CD19 promoter (CD19Cre) (35). Southern blot analysis indicated the highly efficient deletion of caspase-8 gene in purified peripheral B cells from bcasp8-/- mice (Fig. 1A). Western blot analysis of serial dilutions of protein extracts prepared from purified B cells confirmed the loss of caspase-8 protein in bcasp8-/- cells (Fig. 1B).

Analysis by flow cytometry of cell subpopulations in bone marrow (BM), spleen, and lymph nodes (LN) did not show any differences between bcasp8-/- mice and control littermates (Fig. 1C and data not shown). The proportion of T and B cells in LN and spleens and total lymphocyte numbers in thymus, spleen, LN, and BM were similar in control and bcasp8-/- mice (Fig. 1D and data not shown). Therefore, unlike its function in the T-cell lineage, caspase-8 is not essential for the maintenance of B-lymphocyte homeostasis.


Figure 1
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  		FIGURE 1.
Caspase-8 is dispensable for B-cell homeostasis but required for Fas killing. A, Southern blot analysis of caspase-8 deletion in B cells. Caspase-8 floxed allele (casp8fl) and mutant caspase-8 allele lacking exons 3 and4(casp8{Delta}) are indicated. B, Western blot of caspase-8 and actin (loading control) using 2-fold serial dilutions of cell extracts from purified B cells from control and casp8fl/fl CD19-Cre (bcasp8-/-) mice. C, FACS analysis of B lymphocytes populations in spleen (Spl) and LN of bcasp8-/- and control mice. D, cell numbers in thymus, Spl, LN, and BM. Data are presented as the mean ± S.D. of 12 mice for each genotype (p values are 0.32 for thymus, 0.48 for spleen, 0.7 for LN, and 0.5 for BM). E, Fas ligand (FasL) induced cell death in control and bcasp8-/- B lymphocytes. The percent of surviving fraction is shown. UT, untreated; CHX, cycloheximide.

 
To confirm the functional deletion of caspase-8, we assessed the sensitivity of casp8-/- B cells to CD95-induced apoptosis. Purified peripheral B cells were activated in vitro for 3 days with anti-IgM/IL4, and then treated with Fas ligand, in the presence or absence of cycloheximide, an inhibitor of protein biosynthesis known to potentiate Fas killing (37). Annexin-V/PI staining demonstrated that while B cells from control mice were sensitive to CD95-induced killing, bcasp8-/- cells were resistant (Fig. 1E). These results confirm the essential role for caspase-8 in DR-mediated cell death.

Vesicular stomatitis virus (VSV) infection in mice induces a primary infection phase, which consists of a T-cell-independent response characterized by the production of neutralizing IgM antibodies by B cells. The second phase of the infection, which is T-cell-dependent, leads to the production of IgG immunoglobulin (36, 38). We investigated the role of caspase-8 in the in vivo B-cell response to VSV infection. Control and bcasp8-/- mice were subjected to intravenous immunization with 2 x 106 PFU of VSV, and the production of VSV-neutralizing IgM and IgG antibodies was measured 4, 8, and 12 days later (39). While the levels of IgM antibodies directed against VSV were not affected in bcasp8-/- mice, the production of VSV-neutralizing IgG was reduced in bcasp8-/- mice (Fig. 2). This finding is consistent with the increased susceptibility of human patients deficient for caspase-8 to viral infections (22). These findings suggest that caspase-8 deficiency in B cells leads to impaired in vivo responses to VSV infection.


Figure 2
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  		FIGURE 2.
Impaired immunoglobulin production in bcasp8-/- mice following VSV infection. Levels of anti-VSV neutralizing IgM and IgG production are shown at days 4, 8, and 12 post-VSV infection of bcasp8-/- and control mice. p values are 0.1 for IgM at day 4, 0.01 for IgG at day 8, and 0.02 for IgG at day 12 post-infection.

 
B cells are essential components of the innate immune response against microbial infection (24). TLR signaling is important for the activation of B cells and other components of the immune response by microbes (40). Given the connection between DISC components and TLR signaling (29–32), we investigated the effect of caspase-8 deficiency on TLR signaling. This was accomplished by treating purified B-cell populations with antigens specific for different TLRs. B-cell expansion was measured by [3H]thymidine incorporation following treatment with increasing doses of different Escherichia coli-derived LPS, ultrapure LPS, CpG, LTA, or poly (I:C) to identify TLR4, TLR9, TLR2, and TLR3 responses, respectively. Whereas the stimulation by CpG consistently produced similar B-cell expansion in the control and bcasp8-/- backgrounds, responses of casp8-/- B cells to poly(I:C), LPS (Fig. 3A and supplemental Fig. S1), and LTA (supplemental Fig. S2) were reduced compared with controls, demonstrating that caspase-8 does indeed play a role in TLR2, TLR3, and TLR4 signaling. Similarly, CFSE labeling indicated defective expansion of casp8-/- B cells in response to LPS and poly(I:C) stimulation (Fig. 3B and data not shown). These defects could not be attributed to reduce TLR3 and TLR4 protein levels in bcaps8-/- cells as their cell surface expression levels comparable to controls (data not shown). No major defect in proliferation of bcasp8-/- B cells was observed in response to anti-IgM, anti-IgM, and IL4, or anti-IgM and anti-CD40 compared with wild-type controls (data not shown).


Figure 3
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  		FIGURE 3.
Impaired TRL3- and TLR4-mediated B-cell activation in the absence of caspase-8. A, reduced B-cell expansion of bcasp8-/- following LPS (TLR4) poly(I:C) (TLR3), but not CpG (TLR9) stimulation. Cell expansion was determined by measurement of [3H]thymidine incorporation by purified B cells following LPS (*, 0111:B4; **, 055:B5), poly(I:C), and CpG activation. B, kinetics of CFSE loss following LPS activation. CFSE levels in bcasp8-/- and control B cells were evaluated by FACS at 24, 48, and 72 h following LPS activation. Percents of viable and dividing cells post-LPS stimulation are indicated.

 
Cell cycle and cell death were analyzed in response to TLR4 stimulation to characterize the lack of expansion of LPS-stimulated casp8-/- B cells. Based on the PI-stained cell cycle profiles and the quantification of the sub-G1 population, the defective expansion of casp8-/- B cells in response to TLR4 stimulation was attributed to increased cell death (Fig. 4A). Annexin-V/PI staining experiments were also performed and indicated that the increased cell death of TLR4 stimulated casp8-/- B cells was attributed to apoptosis (Fig. 4B). Apoptotic cell death was further confirmed by electron microscopy (Fig. 4, C–F) and increased caspase-3 processing in LPS-stimulated casp8-/- B cells compared with controls (data not shown). Together, these data suggest that caspase-8 is required for the activation of B cells and maintaining their survival in response to TLR3 and TLR4 stimulation.

Upon ligand binding, TLRs initiate signaling cascades that culminate in nuclear translocation of NF{kappa}B, and transcriptional activation of NF{kappa}B target genes (41–43). NF{kappa}B protein members include a family of Rel domain-containing proteins; e.g. p65 (Rel A), Rel B, c-Rel, p50 (NF-B1), and p52 (NF-B2) (44). Activation and nuclear translocation of NF{kappa}B result in increased transcription of its target genes such as TNF-{alpha}, IL6, IL1-{alpha}, IL12p40, and INF-beta (45).

The IKK complex, composed of IKK{alpha}, beta, and {gamma} or NEMO plays an important role in mediating NF{kappa}B nuclear translocation in response to TLR (46). We therefore tested whether caspase-8 associates to this complex upon TLR4 activation. Immunoprecipitation was performed using anti-IKK{alpha}beta and/or anti-caspase-8 antibodies on protein extracts derived from untreated or LPS-stimulated casp8-/- or control B cells. These studies indicated that caspase-8 co-immunoprecipitates with IKK{alpha}beta following 30 min of LPS stimulation of control B cells (Fig. 5A). The specificity of the IKK{alpha}beta immunoprecipitations was shown by Western blot using anti-IKK{alpha}beta antibodies. The recruitment of caspase-8 to a complex containing IKK{alpha}beta in response to LPS stimulation was confirmed in 3T3-immortalized fibroblasts. As shown in Fig. 5B, IKK{alpha}beta association to caspase-8 in 3T3 was detected as early as 4 min following TLR4 activation and appeared to be optimal at 7-min post-LPS stimulation. Thus, our data identify that TLR4 stimulation drives caspase-8 transient recruitment to IKK{alpha}beta, an important regulator of NF{kappa}B functions.


Figure 4
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  		FIGURE 4.
Increased apoptosis of caspase-8-deficient B cells in response to LPS stimulation. A, cell cycle progression and sub-G1 populations were determined using propidium iodide staining and FACS analysis of LPS-stimulated (10 µg/ml) casp8-/- and control B cells. Increased cell death but no defects in cell cycle were observed. B, increased apoptosis in casp8-/- B-cells in response to LPS stimulation (10 µg/ml). Analysis was performed by FACS using Annexin-V/propidium iodide staining. C–F, electron microscopy of casp8-/- B cells before (C and D) or 24-h post-stimulation with LPS (E and F). Nuclear condensation, a feature of apoptosis is seen in E and F.

 
NF{kappa}B is an important downstream target of IKK{alpha}beta and its activation, in response to TLR4 stimulation, leads to transcriptional activation of a subset of target genes. Thus, the effect of caspase-8 mutation on TLR4-induced NF{kappa}B transcriptional activation was assessed using real time PCR to quantify the transcription levels of the NF{kappa}B target genes including IL6, TNF-{alpha}, IFN-beta, and IP-10. cDNA, representative of total cellular mRNA, was prepared from cell-sorted control and casp8-/- B cells activated with LPS for 0, 2, 4, and 7 h. As expected, increased expression of these genes was observed in B cells after treatment with LPS; however, the level of transcriptional induction of these genes over control was significantly lower in casp8-/- B cells (Fig. 5C). Therefore, caspase-8 is important for NF{kappa}B transcriptional activation in response to TLR4-stimulated B cells.


Figure 5
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  		FIGURE 5.
A role for caspase-8 in NF{kappa}B signaling. A, caspase-8 is transiently recruited to IKK complex following LPS/TLR4 stimulation. Immunoprecipitation using anti-IKK{alpha}beta antibodies and cell extracts derived from casp8-/- and control B cells activated with LPS (10 µg/ml) for 5, 15, 30, and 60 min. Co-immunoprecipitation of caspase-8 and IKK{alpha}beta was revealed by Western blot analysis using anti-caspase-8 (upper panel) and anti-IKK{alpha}beta antibodies (lower panel). Total cell extract (+) and an irrelevant polyclonal antibody (-) were used as controls. B, recruitment of caspase-8 to IKK{alpha}beta complex in LPS-stimulated 3T3 fibroblasts. Association of IKK{alpha}beta with caspase-8 in 3T3 cell extracts prepared after 0, 3, 4, 5, 7, 8, and 10 min of LPS treatment was revealed by anti-IKK{alpha}beta Western blot on anti-caspase-8 immunoprecipitates (upper panel). The level of caspase-8 protein was assessed by Western blot analysis using anti-caspase-8 antibodies (lower panel). Total cell extract (+) was used as a control. C, defective transcriptional activation of NF{kappa}B target genes in bcasp8-/- B cells in response to LPS stimulation. Real time PCR was performed on cDNA derived from purified control and bcasp8-/- B cells activated by LPS for the indicated times. The expression of IL6, IFN-beta, TNF-{alpha}, and IP-10 were assessed.

 
To investigate the mechanisms leading to the defective NF{kappa}B-mediated responses of TLR4-stimulated casp8-/- B cells, we focused on known signaling events downstream of LPS/TLR4 activation, including Akt, MAPK-p38, and IkB{alpha} phosphorylation. LPS stimulation of control and casp8-/- B cells for 30 min, 1 h, and 2 h showed comparable levels of expression and phosphorylation of Akt and MAPK-p38 (Fig. 6A and data not shown). Phosphorylation of I{kappa}B{alpha} results in its proteasome-mediated degradation followed by nuclear translocation and transcriptional activation of NF{kappa}B (44). Expression levels and phosphorylation of IkB{alpha} were similar in casp8-/- and control B cells at all time points after LPS stimulation (Fig. 6A).

To further investigate the mechanisms for the defective TLR4-induced NF{kappa}B transcriptional activation in casp8-/- B cells, nuclear translocation of the NF{kappa}B-p65 subunit was assessed by immunofluorescence using control and casp8-/- B cells stimulated with LPS for 0, 30, 45, 60, and 120 min. Similar to controls, NF{kappa}B-p65 was almost exclusively cytoplasmic in untreated casp8-/- B cells (Fig. 6, B and C). However, LPS-induced NF{kappa}B-p65 nuclear translocation was delayed in casp8-/- B cells at 30, 45, 60, and 120 min post-LPS treatment relative to controls (Fig. 6, B and C). Similarly, Western blot analysis of cytoplasmic and nuclear fractions from bcasp8-/- B cells and their controls confirmed a delayed LPS-induced nuclear translocation of NF{kappa}B-p65 in the absence of caspase-8 (Fig. 6D). Similar delayed NF{kappa}B-p65 nuclear translocation was observed in bcasp8-/- B cells in response to TLR3 stimulation (supplemental Fig. S3).

Phosphorylation of NF{kappa}B-p65 at serine 536 is known to play a major role in its nuclear translocation and transactivation (34), and therefore we assessed the effect of loss of caspase-8 on this NF{kappa}B-p65 phosphorylation. Interestingly, the level of phosphorylated NF{kappa}B-p65 at serine 536 was reduced in both cytoplasmic and nuclear fractions from LPS-stimulated bcasp8-/- B cells compared with controls (Fig. 6D). Therefore defective phosphorylation of NF{kappa}B-p65 at serine 536 could potentially contribute to the NF{kappa}B-impaired shuttling and function in LPS-stimulated casp8-/- B cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
CASPASE-8 deficiency has been recently associated with human diseases. These diseases include immunodeficiency, cancer, and metastasis (22). Characterization of CASPASE-8 function in immunodeficient patients has demonstrated that in addition to its apoptotic functions, caspase-8 also carries important non-apoptotic functions.

In this study, we report important roles for caspase-8 in the regulation of NF{kappa}B function in response to TLR4 stimulation. We have previously reported that caspase-8 is dispensable for thymocyte development but required for peripheral T-cell homeostasis and T-cell survival in response to activation stimuli (20). Deletion of caspase-8 in early lymphoid progenitors was also reported to cause early differentiation arrest indicating its important role in early hematopoietic development (21). Similarly, deletion of caspase-8 in all hematopoietic cells using Vav-iCre transgenic mice that express Cre in early hematopoietic progenitors including T- and B-cell progenitors, results in embryonic lethality.4

Our present study demonstrates that CD19-Cre-mediated caspase-8 deletion in B lymphocytes does not affect B-cell development or homeostasis: B-lymphocyte subpopulations were similar in bcasp8-/- mice compared with controls. We also identified defective in vivo production of neutralizing antibodies, one of the main functions of B cells, in the absence of caspase-8 in B cells. Because helper T cells in bcasp8-/- mice express caspase-8 and are fully functional, the deficient immunoglobulin production following VSV infection of bcasp8-/- mice is likely a result of defects in (i) T/B-cell interactions, (ii) immunoglobulin switch, or (iii) B-cell expansion. Our present study supports a key role for caspase-8 in the immune response of B cells and for the proper production of pathogen-neutralizing antibodies.

Toll-like receptors are important components of innate immunity and provide a line of defense against microbial infections. Stimulation of specific TLRs induces a distinct pattern of gene expression, which leads to antigen-specific acquired immunity and activation of the innate immune response pathway. Caspase-8-interacting proteins such as FADD and c-FLIP, or their downstream responsive molecules such as RIP, have been implicated in the regulation of the signaling by some TLR such as TLR3 and TLR4 (29–32). TRAIL-R, known to induce apoptosis and to form a death signaling complex with FADD, and caspase-8 was reported recently to negatively regulate TLR2, TLR3, and TLR4 signaling in macrophages and BM-derived dendritic cells (47). Our study demonstrates that caspase-8 plays an essential role in B-cell activation and expansion in response to TLR2, TLR3, TLR4, but not TLR9.


Figure 6
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  		FIGURE 6.
Delayed NF{kappa}B nuclear translocation in LPS-stimulated bcasp8-/- B cells. A, Western blots showing the levels of expression and phosphorylation of MAPp38 and IkB{alpha} following LPS stimulation (10 µg/ml) of purified control and bcasp8-/- B cells for 0, 0.5, 1, and 2 h. Levels of expression of casp8 and beta-actin (loading control) are shown. B, confocal microscope representative merged views of the p65 subunit of NF{kappa}B(green) and Hoescht (red) from control and bcasp8-/- B cells stimulated with LPS for 0, 30, 45, 60, and 120 min. Data shown are representative of five independent experiments. C, percent of nuclear NF{kappa}B as measured by anti-p65 nuclear staining as in B. D, Western blot analysis of the levels of NF{kappa}B-p65 in cytoplasmic and nuclear fractions from LPS-stimulated purified B cells from bcasp8-/- and control mice. B cells were stimulated with LPS as in A, and cell fractions prepared and used for Western blots with anti-p65, anti-p65 Ser536, anti-PARP, and anti-actin.

 
In contrast to a recent study by Beisner et al. (48), our data support a role for defective NF{kappa}B signaling in the impaired response to TLR3 and TLR4 stimulation of B cells deficient for caspase-8. A possible explanation for this discrepancy is that we have analyzed the effect of caspase-8 loss on NF{kappa}B-p65 nuclear translocation at earlier time points post-TLR3 and -TLR4 stimulation. However, Beisner et al. have analyzed this effect at much later time points when the delay of NF{kappa}B-p65 nuclear translocation is not obvious anymore. Thus, similar to human B cells from caspase-8 mutant patients (23), loss of caspase-8 in mouse B cells impairs nuclear translocation of NF{kappa}B-p65.

TLR4 stimulations activate two major signaling pathways, both dependent on NF{kappa}B-mediated transactivation of genes encoding pro-inflammatory cytokines and survival molecules, among other immune mediators. One pathway is a MyD88-dependent and involves the sequential activation of IRAK-4, binding of TRAF6 to phospho-IRAK1, activation of AP-1 through the MAP kinase pathway, and NF{kappa}B nuclear translocation (49). This pathway induces the early phase of NF{kappa}B-mediated transactivation of genes including IL6 and TNF-{alpha}. The second LPS-activated pathway is MyD88-independent, and activates the late phase of NF{kappa}B-mediated gene expression, driving the production of IFN-beta and the IFN-inducible gene IP-10 (50).

Our real-time PCR analysis identified that in response to TLR4 stimulation, casp8-/- B cells exhibit defective expression of early and late NF{kappa}B-dependent pro-inflammatory cytokines and other immune mediators. We have also identified a temporal delay in the nuclear translocation of NF{kappa}B-p65 in TLR4-stimulated casp8-/- B cells. Delayed NF{kappa}B nuclear translocation has been shown to compromise NF{kappa}B-mediated gene expression, and cell function upon activation (51). These observations highlight the importance of the delayed nuclear translocation of NF{kappa}Bin casp8-/- B cells, in causing the defective production of inflammatory cytokines upon LPS treatment. Thus, we propose a model whereby caspase-8 is involved in the timely activation of NF{kappa}B downstream signals in response to TLR4 activation. In the absence of caspase-8, NF{kappa}B signaling in response to TLR4 activation is not blocked, but delayed. Nevertheless, this delay is sufficient to impair the transduction of NF{kappa}B survival signals in TLR4-stimulated caspase-8-deficient B cells.

Interestingly, we have also identified that in response to TLR4 stimulation, caspase-8 transiently associates with the IKK{alpha}beta, proteins important for NF{kappa}B signaling (52). The IKK complex is responsible for the phosphorylation of various substrates including NF{kappa}B-p65 in response to various stimuli including pro-inflammatory signals (52). Interestingly, we have identified that in response to LPS/TLR4 stimulation, caspase-8 transiently associates with the IKK complex. We also demonstrate that caspase-8 deficiency in TLR4-stimulated caspase-8-deficient B cells leads to decreased phosphorylation of NF{kappa}B-p65 at serine 536. This phosphorylation is mediated by the IKK complex, modulates NF{kappa}B nuclear translocation, and therefore, its impairment could contribute to the defective NF{kappa}B nuclear translocation and functions observed in TLR4-stimulated bcasp8-/- cells. Our findings support a role for defective NF{kappa}B signaling in the impaired responses to TLR4 activation in the absence of caspase-8 and provide a molecular mechanism for the immunodeficiency of caspase-8 mutant patients.


   FOOTNOTES
 
* This work was supported in part by Canadian Institute of Health Research Grant MOP 36537 and National Cancer Institute of Canada Grant TFPP 12000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3. Back

1 To whom correspondence may be addressed: Ontario Cancer Inst., Advanced Medical Discovery Inst., 620 University Ave., Suite 706, Toronto, Ontario M5G 2C1, Canada. Tel.: 416-946-2398; Fax: 416-204-2277; E-mail: rhakem{at}uhnres.utoronto.ca. 2 To whom correspondence may be addressed: Ontario Cancer Inst., Advanced Medical Discovery Inst., 620 University Ave., Suite 706, Toronto, Ontario M5G 2C1, Canada. E-mail: ahakem{at}uhnres.utoronto.ca.

3 The abbreviations used are: DR, death receptor; LPS, lipopolysaccharide; FACS, fluorescent-activated cell sorter; VSV, vesicular stomatitis virus; LTA, lipoteichoic acid; DISC, death-inducing signaling complex; FADD, Fas-associated death domain protein; FLIP, FLICE-like inhibitory protein); RIP-1, receptor-interacting protein-1; LN, lymph node; BM, bone marrow; TLR, toll-like receptor; NK, natural killer cells; PFU, plaque-forming units. Back

4 B. Lemmers, data not shown. Back


   ACKNOWLEDGMENTS
 
We thank W. C. Yeh, B. Au, and M. Woo for helpful discussions. We also thank A. Strasser for kindly providing us with the anti-caspase-8 monoclonal antibody.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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