Differential roles of Yersinia outer protein P-mediated inhibition of nuclear factor-kappa B in the induction of cell death in dendritic cells and macrophages

Yersinia outer protein P (YopP) is injected by Yersinia enterocolitica into the cytosol of host cells by a type III secretion system, which causes cell death in macrophages and dendritic cells (DC) (Zhang et al., 2005; Ruckdeschel et al., 2001; Gröbner et al., 2006, 2007a). In DC we have demonstrated that YopP-induced apoptosis is independent of the mitochondrial death pathway (Gröbner et al., 2006) and that LPS can accelerate YopP-dependent DC death (Gröbner et al., 2007a). In macrophages it was hypothesized that YopP (YopJ in Yersinia pseudotuberculosis) potentiates LPS-induced apoptosis by the inhibition of nuclear factor-kappa B (NF-κB) activation (Ruckdeschel et al., 1998, 2001; Haase et al., 2005; Zhou et al., 2005; Zhang & Bliska, 2003). Furthermore, it has been reported that in macrophages the inhibition of mitogen-activated protein kinases (MAPKs) together with NF-κB is necessary for YopP-induced cell death (Zhang et al., 2005). In the present study we analysed the impact of YopP-dependent NF-κB and MAPK inhibition on cell death induction in DC. Cultivation and infection of DC. Immature DC obtained from bone marrow of BALB/c mice (Harlan Winkelmann, Germany) (Gröbner et al., 2006) or J774A.1 macrophages (ATCC TIB67) were infected with a Y. enterocolitica wild-type strain (pYV⁺) (Heesemann & Laufs, 1983) or a YopP-deficient mutant (yopP) (Ruckdeschel et al., 2001). For this purpose overnight bacteria cultures grown at 27 °C were diluted 1 : 20 in fresh Luria–Bertani broth and grown for a further 1.5 h at 37 °C. The bacterial concentration was measured densitometrically at 600 nm. At 1 h post-infection, at a m.o.i. of 10 : 1, bacteria were killed by gentamicin (100 µg ml^–1) (Sigma). LPS was from Salmonella typhimurium (Sigma), the NF-κB inhibitors MG-132, BAY-11-7082 and BAY-11-7085, and the MAPK inhibitors PD98059, SB203580 and SP600125 were all from Calbiochem.

Assessment of cell death by flow cytometry. Propidium iodide (PI) uptake (50 ng ml^–1) (Calbiochem) and the inner mitochondrial transmembrane potential Δψ_m of DC stained by tetramethylrhodamine ethyl ester (40 nM; Molecular Probes) were determined by flow cytometry (FACSCalibur; BD Biosciences) and analysed by WinMDI version 2.8 software (J. Trotter, The Scripps Institute, La Jolla, CA, USA).

Confocal laser scanning microscopy (CLSM). DC or J774A.1 macrophages were seeded on coverslips and then incubated for 1 h with or without the NF-κB inhibitors MG-132, BAY-11-7082 or BAY-11-7085. Subsequently, LPS was added for 1 h. After fixation, permeabilization and blocking with 100 % inactivated fetal calf serum (Sigma), samples were incubated with rabbit polyclonal p65 antibodies (sc-109; Santa Cruz) 1 : 50 in PBS for 60 min. After three washing steps with PBS, samples were incubated with secondary goat-anti-rabbit-Cy3 antibodies (Dianova) diluted 1 : 100 in PBS for 60 min. After three further washing steps with PBS, nuclei were stained with DAPI (Sigma; 1 µg ml^–1) for 5 min and samples were analysed by CLSM using a Leica TCS SP2 (x63 objective). The images were subsequently processed in Photoshop 7.0.

NF-κB ELISA. A total of 3x10⁷ DC were lysed by MES buffer containing 0.2 mg digitonin ml^–1 as described previously (Gröbner et al., 2006), and the pellets obtained after centrifugation, which contained nuclei and mitochondria, were used for NF-κB ELISAs as recommended in the manufacturer's instructions (NF-κB p65-kit and NF-κB p50 kit; BD Clontech).

Immunoblot analysis. Extracts from 2x10⁶ DC were separated by SDS-PAGE and electrotransferred to nitrocellulose membranes (Schleicher & Schüll). Immunoblot analysis was performed with phospho-specific p38 MAPK antibodies (1 : 2500 dilution; BD) and (anti-mouse) peroxidase-conjugated secondary antibodies (1 : 1000 dilution; Dako) (Fig. 3a). As a loading control, membranes were stripped and reprobed with purified mouse anti-p38α (1 : 1000 dilution; BD) and mouse peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized by using enhanced chemoluminescence reagents (Amersham Biosciences).

(35K): Fig. 3. Inhibition of MAPKs restores the cell death-inducing capacity of a YopP-deficient Yersinia mutant in DC. DC from BALB/c mice or J774A.1 macrophages were preincubated for 30 min with the MAPK inhibitors PD98059 (20 µM), SB203580 (10 µM) and SP600125 (20 µM), which inhibit MEK1/2, p38 and JNK, respectively, and then were infected with Yersinia (pYV+ or yopP). For the flow cytometric analysis extracellular bacteria were killed by the addition of gentamicin (100 µg ml–1) 1 h post-infection and DC (a) or J774A.1 (c) were stained with PI 4 h post-infection. Data in (a) and (c) are mean values±SEM of three individual experiments. *, P<0.01; when compared to the correspondent uninfected cells (DMSO); medium, uninfected cells. (b) Phospho-p38 immunoblot analysis from DC treated for 30 min with SB203580 (10 µM), SP600125 (20 µM) or BAY-11-7082 (10 µM) prior to infection with Yersinia (pYV+, yopP). White bars, Medium; hatched bars, pYV+; black bars, yopP.

Statistical analysis. The two-tailed Student's t-test was used to evaluate the difference in means between two groups. We analysed the differences in YopP-mediated cell death induction between DC and macrophages. LPS was reported to promote YopP-induced cell death in both these cell types. In DC, LPS contributes to YopP-induced cell death in the early phase of Yersinia infection by cleavage of caspase 8 (Gröbner et al., 2007a). In J774A.1 macrophages, LPS potentiates apoptosis induced by the YopP-mediated inhibition of NF-κB activation (Ruckdeschel et al., 1998, 2001; Haase et al., 2005; Zhou et al., 2005; Zhang & Bliska, 2003).

Up to 60 min after infection with Yersinia wild-type, NF-κB translocates to the nucleus of DC due to the LPS of Yersinia, whereas YopP does not inhibit NF-κB activation until 90 min after infection (Erfurth et al., 2004). However, it has been unclear, whether the inhibition of the NF-κB signalling pathway is causative for YopP-induced DC death as has been described in macrophages. To analyse the role of YopP-dependent inhibition of NF-κB activation in the induction of DC death, DC were infected with the Yersinia wild-type strain (pYV⁺) or the YopP-deficient mutant (yopP). As shown in Fig. 1(a), the mitochondrial transmembrane potential already was decreased 90 min after infection with YopP-secreting Yersinia (pYV⁺), but not after infection with YopP-deficient Yersinia (yopP) confirming the findings that Yersinia-induced DC death is caused by YopP (Erfurth et al., 2004; Gröbner et al., 2006). Accordingly, the percentages of PI-positive DC, indicating the lack of cellular membrane integrity, increased as early as 90 min post-infection with pYV⁺ (Fig. 1b). In summary, these results suggest a simultaneous onset of YopP-dependent suppression of NF-κB activation and induction of DC death.

(20K): Fig. 1. YopP of Y. enterocolitica rapidly induces death of DC. DC from BALB/c mice were infected with the Yersinia wild-type strain (pYV+) or the YopP-deficient mutant (yopP). (a) Flow cytometric analysis of the inner mitochondrial transmembrane potential (Δψm) of DC stained with tetramethylrhodamine ethyl ester is shown. (b) Flow cytometric analysis of DC stained with PI is shown. Data are mean values±SEM of three individual experiments. *, P<0.02; **, P<0.002; when compared to yopP-infected cells; medium, untreated cells. Staurosporine (stauro, 4 µM) was used as a positive control.

To further assess whether the inhibition of NF-κB induces death of DC in comparison to macrophages, DC or J774A.1 were preincubated with or without the proteasome inhibitor MG-132, which prevents degradation of the NF-κB inhibitor IkBα, or the NF-κB inhibitors BAY-11-7082 or BAY-11-7085 (Nakanishi & Toi, 2005), and then treated with LPS for 1 h. As shown by Fig. 2(a), LPS caused the translocation of the NF-κB p65 subunit from the cytosol into the nuclei, indicating the activation of NF-κB in both DC and J774A.1. Preincubation of cells with the NF-κB inhibitors distinctly inhibited the LPS-induced nuclear translocation of NF-κB p65, demonstrating the functionality of these inhibitors in DC and J774A.1. As DC spread out when cultivated on poly-L-lysine-coated coverslips, they appeared much larger than J774A.1 macrophages even though the same magnification was used. Additionally, NF-κB activation in DC was quantified by NF-κB ELISA. As shown in Fig. 2(b), the amount of NF-κB p65 in nuclear extracts after stimulation of DC with LPS was about threefold higher compared to DC kept in medium, confirming the results shown in Fig. 2(a). The NF-κB p50 subunit was unresponsive to LPS stimulation (Fig. 2b) confirming the results of a previous study performed in DC (Erfurth et al., 2004). In nuclear extracts of DC infected for 1 h with pYV⁺ or yopP Yersinia the NF-κB amount had doubled within the early phase (≤60 min) of infection suggesting that NF-κB p65 nuclear translocation was caused by bacterial LPS (Fig. 2c), which is in accordance with our previous study (Erfurth et al., 2004). However, when DC had been preincubated with several NF-κB inhibitors 1 h prior to infection, the early Yersinia LPS-induced NF-κB activation was reduced to levels similar to non-infected cells (Fig. 2c), thereby confirming the results shown in Fig. 2(a). Surprisingly, preincubation of DC for 1 h with the NF-κB inhibitors even reduced the percentage of PI-positive cells after infection with Yersinia pYV⁺ for 4 h (Fig. 2d). Hence, it seems unlikely that YopP by inhibition of NF-κB activation contributes to Yersinia-induced DC death. Instead, the reduction of YopP-mediated cell death by different NF-κB inhibitors indicates that there was an inhibition of NF-κB-dependent transactivation of proapoptotic genes, i.e. encoding death receptors, death receptor ligands or proapoptotic members of the Bcl2 family, or alternatively inhibition of the NF-κB-dependent transcriptional repression of anti-apoptotic genes, i.e. encoding TRAF2 (Dutta et al., 2006).

(46K): Fig. 2. LPS-induced NF-κB p65 activation is prevented by NF-κB inhibitors. (a) DC from BALB/c mice or J774A.1 macrophages were seeded on coverslips, preincubated for 1 h with the NF-κB inhibitors MG 132 (10 µM), BAY-11-7082 (10 µM) and BAY-11-7085 (10 µM), and then treated with LPS (1 µg ml–1) for 1 h. After staining of cells with antibodies to NF-κB p65 and Cy3-coupled secondary antibodies, and subsequent staining of nuclei with DAPI (1 µg ml–1), cells were analysed by CLSM. Data are representative of three independent experiments. Bar, 8 µm. (b) After incubation of DC with or without the NF-κB inhibitors, cells were incubated with LPS (1 µg ml–1) for 1 h, and nuclear extracts were analysed by ELISA for the NF-κB p65 and the p50 subunits. Untreated cells (medium) were used as negative controls. Additionally, as an internal negative control, provided by the manufacturer of the analysis kit, nuclear extracts of LPS-treated DC were added to wells coated with a mutant DNA sequence indicating non-specific DNA binding (control). Data are means of two independent experiments performed in duplicate. (c, d) DC or J774A.1 macrophages were preincubated with NF-κB inhibitors as described above and then infected with Y. enterocolitica (pYV+ or yopP). At 1 h post-infection NF-κB p65 ELISA from nuclear extracts were performed as described above (c). Extracellular bacteria were killed by addition of gentamicin (100 µg ml–1) 1 h post-infection and, after staining with PI, cells were analysed by flow cytometry 4 h post-infection (d). Data in (c) and (d) are mean values±SEM of four individual experiments. *, P<0.06; **, P<0.006; when compared to the correspondent DMSO control. Medium, untreated cells. White bars, DMSO; hatched bars, MG-132; grey bars, BAY-11-7082; black bars, BAY-11-7085.

By contrast, cell death of J774A.1 macrophages was not reduced by pretreatment with NF-κB inhibitors. In J774A.1 cells, treatment with MG-132 even increased the cell death rates after pYV⁺-infection. Furthermore, all inhibitors partly restored the cell death-inducing capacity of the yopP mutant in J774A.1 cells, indicating that the inhibition of NF-κB activation is essential for the LPS-triggered induction of cell death in macrophages and confirming the results of former studies (Zhang et al., 2005; Ruckdeschel et al., 1998, 2001). When compared to macrophages only the treatment of DC with MG-132 prior to infection with yopP slightly increased cell death (Fig. 2d), whereas NF-κB specific inhibitors did not. However, in DC infected with pYV⁺ the inhibitory effect of proteasome inhibition by MG-132 on YopP-induced cell death prevailed. Hence, the increase in cell death upon MG-132 treatment in yopP-infected DC might be triggered by reactive oxygen species, which recently have been reported to be overproduced in response to proteasome inhibition (Papa et al., 2007). In summary, our data suggest, that in contrast to J774A.1 macrophages, the inhibition of NF-κB activation is not essential for the induction of YopP-mediated DC death.

It has been reported that the YopP-mediated inhibition of the NF-κB and the MAPK signalling pathways cooperate to promote cell death of macrophages (Zhang et al., 2005). Therefore, we studied whether YopP-dependent DC death results from the inhibition of MAPK activity rather than the inhibition of NF-κB activation. For this purpose, DC or J774A.1 were preincubated with several MAPK inhibitors and subsequently infected with Y. enterocolitica. As shown in Fig. 3(a), preincubation of DC with a p38 inhibitor (SB203580), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125) or with a combination of these inhibitors, but not inhibition of the ERK signalling pathway (PD98059), partly, or even completely, restored the cell death-inducing capacity of the YopP-deficient mutant yopP. To confirm the inhibition of the p38-phosphorylation by YopP in pYV⁺-infected DC and by the p38 specific inhibitor SB203580 in yopP-infected cells, DC were treated with or without SB203580 for 30 min and subsequently infected with Yersinia. As shown by immunoblotting (Fig. 3b), phosphorylation of p38 was inhibited 15, 30 and 45 min post-infection with pYV⁺ (lanes 5, 9 and 13) when compared to DC infected with yopP (lanes 15, 19 and 23). By contrast, phosphorylation of p38 was detected in DC 30 and 45 min post-infection with the yopP mutant (lanes 19 and 23). Preincubation of cells with the p38 specific inhibitor SB203580 caused inhibition of p38 phosphorylation (lanes 20 and 24) in yopP-infected DC, whereas the JNK inhibitor SP600125 (lanes 21 and 25) or the NF-κB inhibitor BAY-11-7082 (lanes 22 and 26), which were both used as controls, did not.

Our data suggest that in Y. enterocolitica YopP-dependent inhibition of MAPK kinases (MKKs) and bacterial LPS cooperate to induce cell death in DC. In particular, inhibitors for the MAPKs p38 or JNK, which are involved in the TNFR-signalling pathway, were sufficient to induce cell death in DC infected with the YopP-deficient strain yopP. Two recent studies describe the induction of apoptosis in cells treated with inhibitors of MAPK and tumour necrosis factor alpha (Furusu et al., 2007; Ricote et al., 2006).

By contrast, inhibition of p38, JNK and MEK1/2 MAPK in macrophages did not restore the cell death-inducing capacities of the yopP mutant. This is in accordance with a previous study performed in J774A.1 macrophages demonstrating that inhibition of MAPK contributes to Yersinia-induced cell death only in cells in which NF-κB was inhibited (Zhang et al., 2005).

Recently, it has been shown that YopJ of Y. pseudotuberculosis, which is homologous to YopP of Y. enterocolitica, catalyses serine/threonine acetylation, thereby mediating the inhibition of MKKs, i.e. MKK6, and the inhibition of NF-κB signalling pathways (Mukherjee et al., 2006; Mittal et al., 2006; Bliska, 2006).

In the present study we provide evidence that YopP induces DC death by the inhibition of MAPKs, predominantly p38 and JNK. Moreover, we have recently demonstrated that YopP promotes the formation of the death inducing signalling complex (DISC), thereby causing the activation of caspase 8 (Gröbner et al., 2007b). Hence, we speculate that YopP might promote the activation of caspase 8 at the level of DISC by acetylating MKK. This idea is supported by a study by Yeh et al. (1998) showing that MKK1 prevents apoptosis at this level.

DC are the most potent antigen-presenting cells. They have the unique ability to prime naive T cells and generate primary immune responses (Banchereau & Steinman, 1998). YopP of Y. enterocolitica induces cell death of mouse DC and thereby prevents DC from inducing T cell adaptive responses (Erfurth et al., 2004). We observed that YopP involves caspase-dependent and, in contrast to macrophages, caspase-independent mechanisms of cell death induction in DC (Gröbner et al., 2006; Ruckdeschel et al., 2002). Moreover, we have shown in this study that in DC, in contrast to macrophages, YopP induces cell death by inhibiting MAPK activity.

This study supports the observation that there are differences between DC and macrophages in the mechanisms of cell death induction by YopP, which might be crucial for the function of these antigen-presenting cells. For example, in DC, which undergo terminal differentiation when infected with Gram-negative bacteria carrying LPS, the involvement of the inhibition of MAPK pathways by Yersinia YopP might allow the bacteria to limit the duration of DC activation and thereby subvert the immune response.

The authors thank Sebastian Wesselborg (Tübingen) for constructive discussion and advice. This work was supported by grants of the Eberhard Karls, University of Tübingen [fortüne 1109, IZKF 1459 (Medizinische Fakultät)], and by the DFG (AU 102).