Tristetraprolin regulates necroptosis during tonic Toll-like receptor 4 (TLR4) signaling in murine macrophages

The necrosome is a protein complex required for signaling in cells that results in necroptosis, which is also dependent on tumor necrosis factor receptor (TNF-R) signaling. TNFα promotes necroptosis, and its expression is facilitated by mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) but is inhibited by the RNA-binding protein tristetraprolin (TTP, encoded by the Zfp36 gene). We have stimulated murine macrophages from WT, MyD88−/−, Trif−/−, MyD88−/−Trif−/−, MK2−/−, and Zfp36−/− mice with graded doses of lipopolysaccharide (LPS) and various inhibitors to evaluate the role of various genes in Toll-like receptor 4 (TLR4)–induced necroptosis. Necrosome signaling, cytokine production, and cell death were evaluated by immunoblotting, ELISA, and cell death assays, respectively. We observed that during TLR4 signaling, necrosome activation is mediated through the adaptor proteins MyD88 and TRIF, and this is inhibited by MK2. In the absence of MK2-mediated necrosome activation, lipopolysaccharide-induced TNFα expression was drastically reduced, but MK2-deficient cells became highly sensitive to necroptosis even at low TNFα levels. In contrast, during tonic TLR4 signaling, WT cells did not undergo necroptosis, even when MK2 was disabled. Of note, necroptosis occurred only in the absence of TTP and was mediated by the expression of TNFα and activation of JUN N-terminal kinase (JNK). These results reveal that TTP plays an important role in inhibiting TNFα/JNK-induced necrosome signaling and resultant cytotoxicity.

The necrosome is a protein complex required for signaling in cells that results in necroptosis, which is also dependent on tumor necrosis factor receptor (TNF-R) signaling. TNF␣ promotes necroptosis, and its expression is facilitated by mitogenactivated protein (MAP) kinase-activated protein kinase 2 (MK2) but is inhibited by the RNA-binding protein tristetraprolin (TTP, encoded by the Zfp36 gene). We have stimulated murine macrophages from WT, MyD88 ؊/؊ , Trif ؊/؊ , MyD88 ؊/؊ Trif ؊/؊ , MK2 ؊/؊ , and Zfp36 ؊/؊ mice with graded doses of lipopolysaccharide (LPS) and various inhibitors to evaluate the role of various genes in Toll-like receptor 4 (TLR4)-induced necroptosis. Necrosome signaling, cytokine production, and cell death were evaluated by immunoblotting, ELISA, and cell death assays, respectively. We observed that during TLR4 signaling, necrosome activation is mediated through the adaptor proteins MyD88 and TRIF, and this is inhibited by MK2. In the absence of MK2-mediated necrosome activation, lipopolysaccharide-induced TNF␣ expression was drastically reduced, but MK2-deficient cells became highly sensitive to necroptosis even at low TNF␣ levels. In contrast, during tonic TLR4 signaling, WT cells did not undergo necroptosis, even when MK2 was disabled. Of note, necroptosis occurred only in the absence of TTP and was mediated by the expression of TNF␣ and activation of JUN N-terminal kinase (JNK). These results reveal that TTP plays an important role in inhibiting TNF␣/JNK-induced necrosome signaling and resultant cytotoxicity.
Toll-like receptor stimulation results in the activation of NF-B, interferon regulatory factor, and MAPK 2 pathways and consequent expression of inflammatory cytokines and chemokines (1,2). The expression of cytokines such as TNF␣ is modulated through posttranscriptional mechanisms. MAP kinaseactivated protein kinase 2 (MK2) and the RNA-binding protein tristetraprolin (TTP) have opposite impact on TNF␣ expression as Mk2 Ϫ/Ϫ cells express reduced levels of TNF␣ in response to TLR signaling (3), whereas TTP-deficient cells express high levels of TNF␣ that is associated with inflammation and autoimmunity (4,5). The p38 MAPK /MK2 pathway stabilizes TNF␣ mRNA and stimulates its translation in part by inactivating (phosphorylating) TTP, which leads to an impairment in the binding of TTP to the AU-rich elements of TNF␣-mRNA (3,(6)(7)(8)(9)(10).
In addition to TNF-R engagement, TLR signaling in the absence of caspase activity results in the assembly of an necroptosis (22)(23)(24) The pathological role of necroptosis has been revealed in various chronic diseases (25)(26)(27)(28)(29)(30)(31)(32)(33). Recently, it was reported that the treatment of myeloid leukemic cells with SMAC mimetic failed to induce TNF␣ expression and ripoptosome induced cell death unless MK2 was inhibited (34). Furthermore, MK2 was shown to induce an inhibitory phosphorylation at Ser-321 on RipK1 in myeloid leukemic cells that restricted SMAC mimetic-induced cell death (34 -37). However, this enhancement of cell death by the inhibition of MK2 was not dependent on MLKL or RipK3 but required the kinase function of RipK1 (34).
Here we evaluated the impact of MK2 and TTP in macrophages, and we demonstrate that TTP inhibits necrosome activation during tonic TLR signaling through the inhibition of TNF␣ and JNK expression/activation.

MyD88 and TRIF signaling promote necroptosis of macrophages
Necrosome signaling is induced in cells by engagement of TLRs or cytokine receptors (TNF-R/IFNAR) signaling in the absence of caspase-8 activity (17,38). We stimulated macrophages with various concentrations of the TLR4 ligand LPS in the presence of a high concentration of the pan-caspase inhibitor zVAD-fmk (50 M) to identify the relative concentration of LPS required to induce cell death by necroptosis. To confirm the mechanism of cell death as necroptosis, we treated cells with necrostatin-1 (Nec-1) to inhibit the kinase region of RipK1 and consequent RipK1-RipK3 interaction. Our results indicate that a minimal concentration of 200 pg/ml of LPS is required to induce necroptosis of macrophages (Fig. 1A). Furthermore, at 10 ng/ml of LPS, 50% cell death of macrophages occurred by 6 h post stimulation, whereas at 0.1 ng/ml LPS, 50% cell death occurred at 24 h (Fig. 1B). Necroptosis was not induced when . C-J, BMMs were generated from WT or various knockout mice indicated in the figure and treated with LPS (100 ng/ml) and zVAD-fmk (50 M). Cell viability was evaluated by MTT assay (24 h) (C and J), alamarBlue assay (24 h) (D), or at 18 h by Zombie Yellow assay (E). Cell extracts were collected at various time intervals and the expression/activation of various proteins evaluated by Western blotting (F and G). Production of TNF␣ (ELISA) and IFN-I (bioassay) was measured in cell supernatants collected at 24 h (H). A graphic version of results is shown in panel I. Cells were also treated with LPS and zVAD-fmk as mentioned above and p38MAPK inhibitor (LY2228820, 4 M) or MK2 inhibitor (PF3644022, 5 M) (J), and cell death was evaluated at 24 h by MTT assay. Representative data of one experiment of three similar experiments are shown. Graphs show the percentage of viable cells Ϯ S.D. relative to controls. Each experiment was repeated three times. *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001; ****, p Ͻ 0.0001.

TTP regulates necrosome signaling
the concentration of LPS was reduced to 0.01 ng/ml. This suggests that higher concentrations of LPS accelerate the timing of necroptosis. We stimulated primary macrophages with high (100 ng/ml) (Fig. 1, A, C-I) or low (1 ng/ml) (Fig. S1, A-F) concentration of LPS, and/or zVAD-fmk (50 M) and measured cell death at 24 h. Both at high and low concentration of LPS, cell death depended on RipK3 and was rescued by the RipK3 inhibitor GSK872 (Fig. 1C and Fig. S1A). Whereas Ifnar1 Ϫ/Ϫ macrophages showed resistance against necroptosis at both LPS concentrations ( Fig. 1C and Fig. S1A), Tnfr2 Ϫ/Ϫ macrophages showed protection only in response to stimulation with low concentration of LPS (Fig. S1A). Thus, higher concentration of LPS shifts the dependence of cell death less toward TNFR2 and more toward IFN-R. There was no impact of TNFR1 on necroptosis. Although we have previously reported that TRIF-signaling promotes necroptosis of macrophages (38), our results indicate that resistance against necroptosis induced by high or low concentration of LPS occurs only when both MyD88 and TRIF-signaling pathways are disabled ( Fig. 1, D and E and Fig. S1B). Combined deficiency in MyD88 and Trif compromised necrosome signaling in macrophages as revealed by lack of Ser-166 phosphorylation of RipK1 and undetectable Ser-345 phosphorylation of MLKL (Fig. 1F). Furthermore, the total phosphorylation of RipK1 (upper band) was undetectable in MyD88 Ϫ/Ϫ Trif Ϫ/Ϫ macrophages (Fig. 1F). We further observed that the phosphorylation of RipK3 was reduced in Trif Ϫ/Ϫ but completely inhibited in MyD88 Ϫ/Ϫ Trif Ϫ/Ϫ macrophages (Fig. 1G). This suggests that both TLR4 adaptor proteins synergize to induce necroptosis with TRIF playing a more dominant role over MyD88. We observed that MyD88 promoted the expression of TNF␣ whereas TRIF induced IFN-I. The expression of both cytokines was inhibited only in macrophages with double deficiency of TRIF and MyD88 ( Fig. 1H and Fig. S1C).
Interestingly, the activation of p38 MAPK was reduced in MyD88 Ϫ/Ϫ and Trif Ϫ/Ϫ macrophages but was undetectable in MyD88 Ϫ/Ϫ Trif Ϫ/Ϫ macrophages (Fig. 1F). Thus, despite little p38 MAPK phosphorylation in MyD88 Ϫ/Ϫ Trif Ϫ/Ϫ macrophages, there was poor necroptosis of macrophages. This result is surprising because p38 MAPK pathway has been recently shown to induce an inhibitory phosphorylation of RipK1 and inhibit cell death (35)(36)(37). Because we observed that the phosphorylation of RipK1 (Ser-166)-dependent necroptosis was also inhibited in MyD88 Ϫ/Ϫ Trif Ϫ/Ϫ macrophages, this result suggests that the inhibition of the p38 MAPK -induced inhibitory phosphorylation of RipK1 does not necessarily drive the cells toward necroptosis in the absence of TNF␣ and IFN-I.
Because p38 MAPK and the downstream kinase MK2 promotes TNF␣ expression by macrophages following LPS treatment (3), we evaluated the impact of p38 MAPK inhibitor (LY2228820, 4 M) or MK2 inhibitor (PF3644022, 5 M) on necroptosis induced by high (Fig. 1I) or low (Fig. S1D) concentrations of LPS. There was no appreciable increase in necroptosis when the p38 MAPK pathway was disabled. Similar results were observed with Mk2 Ϫ/Ϫ macrophages ( Fig. 1I and Fig. S1E). Total phosphorylation of RipK1 (upper band) was inhibited in Mk2 Ϫ/Ϫ cells and there was a slight increase in the Ser-345 phosphorylation of MLKL at earlier time period (Fig. S1F).

MK2 promotes TNF␣ expression but impairs the sensitivity of cells to necroptosis at lower levels of TNF␣
Whereas induction of necroptosis in macrophages requires high concentrations of zVAD-fmk (Ͼ25 M) ( Fig. 2A), lower concentrations are needed to inhibit caspase activity (39). We therefore revised our experimental model and treated cells with LPS (1 ng/ml) and a reduced concentration of zVAD-fmk (10 M) that does not induce significant necroptosis ( Fig. 2A) to determine whether the inhibition of p38 MAPK induces their sensitivity to cell death under these conditions. Our results indicate that the inhibition of p38 MAPK makes the cells significantly susceptible to cell death (Fig. 2B), which correlates with Ser-345 phosphorylation of MLKL (Fig. 2C). We observed that the inhibition of p38 MAPK accelerated the kinetics and magnitude of necroptosis ( Fig. 2D) compared with the cells that did not receive the p38 MAPK inhibitor (Fig. 1B). Induction of cell death by the inhibition of p38 MAPK was still dependent on RipK3, TNF␣ ( We observed that the expression of TNF␣ was potently reduced in Mk2 Ϫ/Ϫ cells in response to stimulation with LPS ( Fig. S2D) or LPSϩzVAD-fmk (Fig. 2K), although cell death occurring in the absence of MK2 is TNF␣ dependent (Fig. 2G). To reconcile this paradox, we treated Tnfa Ϫ/Ϫ macrophages with LPS (1 ng/ml)ϩzVAD-fmk (10 M) in the presence of increasing concentrations of recombinant TNF␣. Our results indicate that the inhibition of MK2 or p38 MAPK makes macrophages highly sensitive (Ͼ100-fold) to minimal concentrations of TNF␣ required to induce cell death ( Fig. 2L and Fig. S2E). These results suggest that the inhibition of p38 MAPK /MK2 signaling increases the sensitivity of macrophages to necroptosis induced by very low levels of TNF␣.

p38 MAPK restricts necroptosis induced by the inhibition of caspase-8
We observed that when we use higher concentration (50 M) of the pan-caspase inhibitor zVAD-fmk, potent necrop-TTP regulates necrosome signaling tosis is induced in macrophages, and addition of the p38 MAPK inhibitor has negligible impact (Fig. 1J). However, when the concentration of zVAD-fmk is lowered to 10 M, then necroptosis is induced only when the p38 MAPK pathway is inhibited (Fig. 2B). Thus, the use of higher zVAD-fmk concentration masks the important role of p38 MAPK in the inhibition of necrosome signaling. Interestingly, treatment of macrophages with LPS (1 ng/ml) and the specific caspase-8 inhibitor (zIETD-fmk, 50 M) or another pancaspase inhibitor (Q-VD-OPh, 50 M) did not induce necroptosis (Fig. 2M). These results suggest that zVAD-fmk is uniquely able to induce necroptosis. Interestingly, we observed that zIETD-fmk does induce necroptosis of macro-phages only when p38 MAPK is inhibited (Fig. 2, M and N). In addition to zIETD-fmk, treatment of cells with Q-VD-OPh resulted in cell death only upon co-treatment with LPS and p38 MAPK inhibitor (Fig. 2M). Because the zIETD-fmk induced potent necrosome signaling in macrophages only when p38 MAPK /MK2 signaling was inhibited, this suggests that zIETD-fmk was functional as a caspase-8 inhibitor. Furthermore, treatment of macrophages with LPSϩ zIETD-fmkϩp38 MAPK inhibitor resulted in the phosphorylation of MLKL (Fig. 2O).

TTP regulates necrosome signaling
fore used another specific caspase-8 inhibitor, Emricasan, which has increased specificity toward caspase-8 and blocks cFLIP-caspase-8 heterodimers similar to zVAD-fmk (42). The treatment of cells with LPS (1 ng/ml)ϩEmricasan (10 M) resulted in cell death, which was enhanced by co-treatment with p38 MAPK inhibitor (Fig. 2P). Cell death was rescued by GSK872, indicating the mechanism of cell death to be necroptosis. Additional experiments indicated that when the concentration of Emricasan was reduced to 1 M, cell death was undetectable in macrophages unless p38 MAPK pathway was inhibited (Fig. S2F). It has been shown that the phosphorylation of RipK1 by TAK1 governs the induction of apoptosis or necroptosis (43). We observed that the inhibition of TAK1 results in enhanced cell death of macrophages, and that was comparable to what was observed with the p38 MAPK inhibitor (Fig. S2G).

TTP inhibits necrosome activation during tonic TLR4 signaling
Because TTP (Zfp36) is a downstream target of MK2 (MAPKAP2), we evaluated the role of TTP in necroptosis of macrophages. Our results indicate that when Zfp-36 Ϫ/Ϫ macrophages are stimulated with 1 ng/ml of LPS and 25 M zVAD-fmk, they undergo significantly more necroptosis (Fig.  S3). We reasoned that this regulation of necroptosis by TTP may become more apparent at even more reduced concentrations of LPS. We observed that when cells were stimulated with very low dose of LPS (0.01 ng/ml), WT macrophages failed to undergo cell death, whereas Zfp36-deficient macrophages displayed a slight increase in cell death (Fig. 3, A-C). Interestingly, the inhibition of p38 MAPK in WT cells did not induce any detectable cell death at low LPS concentration, whereas the inhibition of p38 MAPK induced significant cell death in Zfp36 Ϫ/Ϫ macrophages (Fig. 3, D-G). This induction of cell death in Zfp36 Ϫ/Ϫ macrophages was because of necroptosis because the inhibition of RipK3 by GSK872 rescued cell death. Interestingly, the potent activation of necrosome, as revealed by Ser-345 phosphorylation of MLKL and Ser-166 phosphorylation of RipK1, was observed only when p38 MAPK pathway was inhibited in Zfp36 Ϫ/Ϫ cells (Fig. 3H).
Because RipK1 has been shown to have cell death-independent role in promoting cytokine synthesis through ERK1/2 and NF-B activation (44), and TTP has been reported to promote degradation of various signaling proteins in the NF-B pathway (45), we evaluated the activation of NF-B by Western blotting. There was an increase in the activation of NF-B in Zfp36 Ϫ/Ϫ macrophages (Fig. 3I). Comparable to the results obtained with the inhibitor of p38 MAPK , the inhibition of MK2 also resulted in cell death in Zfp36 Ϫ/Ϫ cells (Fig. 3J). The inhibition of p38 MAPK pathway also resulted in enhanced cell death in Zfp36 Ϫ/Ϫ cells when cells were treated with low concentration of LPS and the specific inhibitor of caspase-8 (Emricasan) (42) (Fig. 3K). We observed that the inhibition of TAK1 or p38 MAPK resulted in comparable enhancement in the induction of necroptosis in Zfp36 Ϫ/Ϫ macrophages (Fig. S4A). The inhibition of p38 MAPK resulted in enhanced RipK3 phosphorylation in Zfp36 Ϫ/Ϫ macrophages (Fig. S4B).

The inhibition of necrosome signaling by TTP occurs through abrogation of both TNF␣ expression and JNK activation
Because regulation of TNF␣ mRNA stability is critically dependent on TTP (3), we evaluated the impact of TTP and p38 MAPK pathway on TNF␣ expression in response to tonic LPS stimulation. At a highly reduced concentration of LPS (0.01 ng/ml) and zVAD-fmk (10 M), the TNF␣ production was undetectable in WT cells but was significantly induced in Zfp36 Ϫ/Ϫ cells particularly after inactivation of the p38 MAPK pathway (Fig. 4, A and B). Necroptosis of Zfp36 Ϫ/Ϫ macrophages, following stimulation with low level of LPS (10 pg/ml), was partially inhibited by blocking TNFR-I and completely rescued by blocking TNFR-II (Fig. 4, C-F). This suggests that TTP blocks necrosome signaling through regulation of TNFR signaling. Addition of exogenous TNF␣ to cells instead of LPS resulted in induction of necroptosis in both WT cells and Zfp36 Ϫ/Ϫ macrophages to the same degree (Fig. 4G), which correlated with the Ser-166 phosphorylation of RipK1 and Ser-345 phosphorylation of MLKL (Fig. 4H). This is in contrast to the situation with LPS treatment where cell death and necrosome activation was only observed in Zfp36 Ϫ/Ϫ macrophages (Fig. 3, D-H). These results suggest that the difference in cell death observed in WT and Zfp36 Ϫ/Ϫ macrophages following LPS treatment must be due selective induction/maintenance of TNF␣ expression in Zfp36 Ϫ/Ϫ macrophages.
Thus, these results indicate that when necrosome activation is induced by very high concentrations of both LPS and the pan-caspase inhibitor zVAD-fmk, the impact of p38 MAPK /MK2 signaling on necroptosis is barely appreciable. The inhibitory effect of p38 MAPK /MK2 on necrosome signaling becomes apparent only when the concentrations of LPS and zVAD-fmk are reduced to low levels. Because TTP degrades TNF␣ transcripts (3), we predict that this results in complete abrogation of TNF␣ when cells are stimulated with low doses of LPS, and consequently, no necrosome signaling ensues. However, abrogation of TTP is not sufficient to switch the cells to necroptosis because MK2 still promotes the inhibitory phosphorylation of RipK1. Thus, disabling both the TTP and MK2 pathways is required to promote the necrosome activation of macrophages (Fig. 6).

Discussion
TLR signaling of myeloid cells is a key driver of inflammatory response that facilitates the control of pathogens (1). Rupture of cells as a result of inflammatory cell death pathways results in the release of DAMPs and further amplification of the inflammatory response, which can lead to impairment in host survival (28). The triad of RipK1, caspase-8, and RipK3 maintains home-TTP regulates necrosome signaling ostasis, and an imbalance in the expression/function of any one of these proteins leads to host fatality because of over-activation of the others (47)(48)(49)(50). Thus, regulation of cell death pathways is critical for maintenance of homeostasis. Although caspase-8 is considered a bona fide regulator of necrosome signaling, additional regulatory mechanisms of necrosome signaling have been recently revealed (24,(34)(35)(36)(37). In this report we show that TTP plays a key role in inhibiting necrosome signaling of macrophages.
Our results indicate that during necrosome signaling of WT macrophages, p38 MAPK /MK2 signaling does not impact necroptosis of cells unless the concentration of zVAD-fmk is reduced or caspase-8 inhibitor is specifically used. Although caspase-8 is a potent inhibitor of necrosome signaling in fibroblasts, specific inhibition of caspase-8 by zIETD-fmk does not result in necroptosis of macrophages even at high concentrations (24). Although it was considered that the concentration of the caspase-8 inhibitor used (50 M) may not be sufficient to inhibit caspase-8, it inhibited necrosome signaling when p38 MAPK /MK2 signaling was also inhibited, suggesting that the caspase-8 inhibitor is functional at that concentration. It is currently not clear why necroptosis in macrophages can be induced by the pan-caspase inhibitor zVAD-fmk but not by the caspase-8 inhibitor zIETD-fmk (24) or the other pan-caspase inhibitor, Q-VD-OPh. Because cFLIP is an endogenous inhibitor of caspase-8 (40) which interacts with caspase-8 as a het-  erodimer (41), it is conceivable that zVAD-fmk uniquely inhibits the caspase-8 -cFLIPs heterodimer better than the zIETD or Q-VD-fmk.

TTP regulates necrosome signaling
We have reported previously that during necrosome signaling in macrophages, TLR4-engagement induces the phosphorylation of RipK1 at Thr-235 and Ser-313 (51). Recently, a new inhibitory phosphorylation of RipK1 was reported at Ser-321 in myeloid leukemia cells following treatment with SMAC mimetics, and that was mediated by MK2 (34 -37). However, this enhancement of leukemic cell death that was induced by the inhibition of MK2 was not dependent on MLKL or RipK3 but required the kinase function of RipK1 (34). The role of MK2 in necroptosis was evaluated following treatment of MEFs with TNF␣ϩSMAC-mimeticϩzVAD-fmk, and this was reported to be enhanced by the inhibition of MK2 (37). Although these studies did not evaluate the impact of MK2 in TLR4-induced necrosome signaling of macrophages, we did not observe any significant impact of MK2 when cells were stimulated with traditional doses of LPS (1 or 100 ng/ml) and zVAD-fmk (50 M) required to induce necroptosis of macrophages. Rather, we observed that necroptosis was induced only when cells were stimulated with very low doses of LPS (1 ng/ml) and zVAD-fmk (10 M), and the p38 MAPK pathway was inhibited. Furthermore, our results also indicate that the inhibition of the p38 MAPK pathway enhances necroptosis induced by TNF␣ϩzVAD-fmk; however, this was not impacted by Zfp-36 (Fig. 4G). We have revealed that the role of Zfp-36 is mainly related to expression of TNF␣ when TLR4 signaling is at tonic levels. Under these conditions, the inhibition of p38 MAPK pathway by itself fails to induce necroptosis in WT cells (Fig. 3D). We believe that signaling mechanisms that operate at very low levels of stimulants are physiologically more relevant as they may exercise a greater impact on homeostasis.
We have previously reported that the higher molecular weight band of RipK1 is the phosphorylated version of this protein (38). Because this phosphorylation of RipK1 was inhibited in MK2-deficient cells, it appears that the predominant phosphorylation of RipK1 in response to TLR signaling is inhibitory

TTP regulates necrosome signaling
in nature and is mediated by MK2. The knockdown of RipK1 has been shown to enhance necroptosis of human monocytic THP1 cells (52), suggesting that RipK1 scaffold may function more as an inhibitor rather than activator of necroptosis. Interestingly, disabling p38 MAPK /MK2 results in amplification of the stimulatory RipK1 phosphorylation (Ser-166) while completely obliterating the inhibitory phosphorylation, and this dichotomy becomes more pronounced when necrosome stimulation is reduced, particularly when TTP is also disabled. It has been shown that direct phosphorylation of RipK1 at Ser-321 by MK2 suppresses RipK1 Ser-166 autophosphorylation and inhibits its ability to bind FADD/caspase-8 and induce RipK1-kinase-dependent apoptosis and necroptosis (37).
We have reported that IFNAR1 signaling (53), and particularly the downstream transcription factor complex ISGF3, is required for necrosome activation in macrophages (38). Recently, TNF-R2 signaling has been shown to be required for necroptosis of macrophages that depends on IFNAR1 signaling (54). This explains why necrosome signaling depends on IFNAR1 and TNFR2 particularly when a relatively reduced dose of LPS (1 ng/ml) is used. It is not clear why necroptosis becomes less dependent on TNFR2 when the concentration of LPS is increased further to 100 ng/ml. An interesting paradox that we have revealed here is that during necrosome signaling the levels of TNF␣ are substantially reduced in MK2-deficient macrophages, yet these cells become highly susceptible to TNF␣-dependent necroptosis. We have shown that MK2 deficiency results in increased sensitivity of macrophages to necroptosis at highly reduced TNF␣ concentrations.
Our results reveal that the p38 MAPK and TTP pathways synergize to regulate necrosome signaling, and this synergistic regulation of necrosome signaling achieves a greater significance when the concentrations of LPS and zVAD-fmk are reduced. According to our model, TTP inhibits TNF␣ expression when the TLR4 signaling is very low, and TNF␣ is required for necrosome signaling. The inhibition of TTP does not enhance necroptosis because MK2 is still able to induce an inhibitory phosphorylation on RipK1 and hence inhibit necroptosis. Thus, disabling both TTP and MK2 results in necroptosis.
It has been previously reported that transfection of the human embryonic kidney cell line HEK293 with a TTP and XIAP-expressing plasmid results in maintenance of RipK1 expression through degradation of XIAP and cIAP2 causing increased cell death (55). Our Western blots did not reveal any impact of TTP on the expression of RipK1 (Fig. 5, A and B), although we did observe increased expression of cIAP1/2 and XIAP in Zfp36-deficient macrophages. However, IAPs inhibit ripoptosome-induced cell death (15,39), without having any   (1). TNF␣ mRNA is rapidly degraded by TTP through recognition of AU-rich regions (2). As a consequence, less TNF␣ is available for TNF-receptor signaling and activation of JNK1/2 (3). Small levels of TNF␣ bind to the TNF-receptor (4) which is sufficient to induce phosphorylation of TAK1, P38, MK2, and Ser-166 -RipK1 (5). MK2 induces an inhibitory Ser-321 phosphorylation of RipK1 (6). As a consequence, RipK3 fails to phosphorylate MLKL and mediate necroptosis (7). TTP regulates the level of TNF␣ which is the critical first step in promoting the activation of RipK1.

TTP regulates necrosome signaling
impact on necrosome signaling and necroptosis (56). It remains to be seen whether the increased expression of XIAP in Zfp36-deficient macrophages results in reduced ripoptosome-induced cell death following treatment with a SMAC mimetic. In contrast to the effect of RipK1 in ripoptosome signaling, RipK1 has been shown to inhibit necrosome signaling (52). Similar to necrosome signaling, TTP was recently shown to inhibit the expression of NLRP3 inflammasome-mediated cell death (57). Thus, we believe that the regulation of TNF␣ expression by TTP and induction of the inhibitory phosphorylation on RipK1 by MK2 result in synergistic inhibition of necrosome signaling in macrophages (Fig. 6). Both, Zfp-36-and MK2-deficient mice have major phenotypic impact in vivo (3,4). We expect that necrosome signaling is dysregulated and facilitates the effects observed in the inflammatory response in these mice.

Macrophages and cell cultures
Macrophages (BMM) were generated from the bone marrow of the appropriate mouse strain after differentiating bone marrow cells with M-CSF (5 ng/ml) for 7-12 days. Cells were cultured in RPMI ϩ 8% FBS. Cells were plated in 96-or 24-well plates in RPMIϩ8%FBS and various reagents added after overnight incubation. At various time intervals, cell death and activation of various proteins were evaluated.

Cell death assays
Cell viability was measured by various approaches. For colorimetric assay of cell death measurement, MTT was added to cells at a final concentration of 0.5 mg/ml and incubated at 37°C for 2 h. Cells were lysed by adding 5 mM HCl in isopropyl alcohol and vigorously pipette up and down to solubilize the MTT crystals. Using Emax plate reader, optical density values were obtained by measuring absorption at 570 nm with a reference wavelength of 650 nm. The data were normalized to the corresponding stimulated control (i.e. LPSϩzVAD-fmk treated cells were normalized to LPS treated cells whereas TNF␣ϩzVAD-fmk treated cells were normalized to TNF␣). In some experiments cell viability was measured by CCK8 assay (Enzo Life Sciences, Cat#ALX-850 -039-K101). Optical density was measured at 450 nm and normalized to the corresponding stimulation control.
Cell death was also evaluated by immunofluorescence following staining with Hoëchst 33342 (2.5 g/ml; Invitrogen) and propidium iodide (1:10 dilution; BD Pharmingen, 550825). Cells were stained in RPMI lacking phenol for 25-30 min. to distinguish live and dead cells. A Zeiss AxioObserver D1 microscope and the AxioVision Rel. 4.8 program were utilized to capture and analyze images. Cell death was also evaluated by flow cytometry following staining of cells with Zombie Yellow TM (BioLegend, San Diego, CA). Zombie Yellow TM solution (1:100 in PBS) was added into each well and the plate was incubated at room temperature, in the dark, for 15-30 min before washing with 100 l FACS buffer (PBS, 1% BSA, 1 mM EDTA) once. Samples were fixed in 1% paraformaldehyde and acquired on LSR Fortessa Flow cytometer.