Necroptosis Suppresses Inflammation Via Termination of TNF- Or LPS-Induced Cytokine and Chemokine Production

Cell Death and Differentiation (2015) 22, 1313–1327 & 2015 Macmillan Publishers Limited All rights reserved 1350-9047/15 www.nature.com/cdd Necroptosis suppresses inflammation via termination of TNF- or LPS-induced cytokine and chemokine production

CJ Kearney1, SP Cullen1,2, GA Tynan3, CM Henry1, D Clancy1, EC Lavelle2,3 and SJ Martin*,1,2

TNF promotes a regulated form of necrosis, called necroptosis, upon inhibition of caspase activity in cells expressing RIPK3. Because necrosis is generally more pro-inflammatory than apoptosis, it is widely presumed that TNF-induced necroptosis may be detrimental in vivo due to excessive inflammation. However, because TNF is intrinsically highly pro-inflammatory, due to its ability to trigger the production of multiple cytokines and chemokines, rapid cell death via necroptosis may blunt rather than enhance TNF-induced inflammation. Here we show that TNF-induced necroptosis potently suppressed the production of multiple TNF-induced pro-inflammatory factors due to RIPK3-dependent cell death. Similarly, necroptosis also suppressed LPS-induced pro-inflammatory cytokine production. Consistent with these observations, supernatants from TNF-stimulated cells were more pro-inflammatory than those from TNF-induced necroptotic cells in vivo. Thus necroptosis attenuates TNF- and LPS-driven inflammation, which may benefit intracellular pathogens that evoke this mode of cell death by suppressing host immune responses. Cell Death and Differentiation (2015) 22, 1313–1327; doi:10.1038/cdd.2014.222; published online 23 January 2015

Tumor necrosis factor (TNF) receptor (TNFR) stimulation can and secretion of a diverse array of cytokines and chemokines – result in apoptosis via FADD-dependent recruitment of that collectively trigger the inflammatory response.16 18 From caspase-8 to the TNFR intracellular death domain.1,2 Recent this perspective, necroptosis could be interpreted as a studies have also shown that TNFR engagement can result in strategy by viruses encoding caspase inhibitors to shut down a form of programmed necrosis, called necroptosis, where the host pro-inflammatory programme by terminating synth- TNF-induced caspase-8 activation is frustrated either using esis of pro-inflammatory cytokines and chemokines. pharmacological or viral-derived caspase inhibitors,3–5 or as a Key biological outcomes of TNFR stimulation includes the consequence of targeted disruption of the CASP-8 or FADD recruitment of peripheral blood neutrophils through the loci.6–8 Necroptosis is generally viewed as a pro-inflammatory generation of gradients of chemokines such as IL-8/MIP-2, mode of cell death, and many studies have concluded that this recruitment of monocytes/macrophages (via MCP-1), activa- represents a host response to viral infection that limits viral tion of local macrophages and dendritic cells (via MCP-1 and replication.9–11 However, it is frequently overlooked that the GM-CSF), induction of monocyte/macrophage differentiation majority of TNF-responsive cell types do not undergo (GM-CSF), activation of local endothelium,16,19,20 as well apoptosis, or indeed necrosis, in response to TNFR engage- as a range of other responses.16,21 Therefore the central role ment. Instead, most cells initiate highly robust nuclear factor of TNF as a trigger for pro-inflammatory cytokine/chemokine kappa B (NFκB)-dependent pro-inflammatory responses upon production, rather than apoptosis, is a key issue when stimulation with this cytokine. evaluating the outcome of interventions, such as caspase TNF stimulation rapidly results in recruitment of ‘Complex I’ inhibition, that impact on TNF-induced signal transduction components to the TNF receptor, which rapidly signal to NFκB pathways. activation.12 Complex I-mediated NFκB activation restrains Because necrosis is generally pro-inflammatory, in activation of caspase-8 in a cytosolic ‘complex II’.12 To comparison with apoptosis, it is widely presumed that the undergo apoptosis in response to TNF, cells typically require switching of TNF-induced signaling to necroptosis exacer- sensitization through the use of transcriptional/translational bates inflammation.22 The latter view is predicated upon the inhibitors or IAP-antagonists that block NFκB activation idea that necrosis results in the release of intracellular signals.13–15 Thus the primary consequence of TNFR constituents, called damage-associated molecular patterns stimulation in responsive cell types is the rapid synthesis (DAMPs), that promote inflammation.23–26 However, the

1Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin D2, Ireland; 2Immunology Research Centre, Trinity College, Dublin D2, Ireland and 3Adjuvant Research Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College, Dublin D2, Ireland *Corresponding author: SJ Martin, Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute of Genetics, Trinity College, Dublin D2, Ireland. Tel: +353 1 8961289; Fax: +353 1 679 8558; E-mail: [email protected] Abbreviations: TNF, tumor necrosis factor; LPS, lipopolysaccharide; RIPK3, receptor-interacting protein kinase 3; FADD, fas-associating death domain-containing protein; TNFR, TNF receptor; NFκB, nuclear factor kappa B; MCP-1, monocyte chemotactic protein 1; RANTES, regulated upon activation normal t cell expressed and presumably secreted; MIP-2, macrophage inflammatory protein 2; KC, keratinocyte chemoattractant; GMCSF, granulocyte-macrophage colony-stimulating factor; IL-8, interleukin 8; MEF, murine embryonic fibroblast; MLKL, mixed lineage kinase domain-like; Nec1, Necrostatin 1; CrmA, cytokine response modifier A Received 08.9.14; revised 12.11.14; accepted 19.11.14; Edited by G Melino; published online 23.1.15 Necroptosis suppresses inﬂammation CJ Kearney et al 1314

presumption that TNF-induced necroptosis represents a more pro-inflammatory than TNF-initiated outcomes that occur in – inflammatory outcome than TNF stimulation alone fails to take the presence of caspase activity.22,27 29 This view appears to account of the fact that TNF is intrinsically highly probe predicated upon the notion that TNF promotes either inflammatory, as noted earlier. An alternative possibility is that apoptosis or necrosis, with the latter typically being pro- necroptosis may suppress inflammation through abruptly inflammatory and the former not. However, because TNF is terminating TNF-induced cytokine production. It is not known itself highly pro-inflammatory as illustrated above (Figures 1a whether the release of intracellular DAMPs, due to and b), the impact of necroptosis on TNF-induced inflamma- necroptosis-associated cell rupture, compensates for inhibitory events remains unclear. As shown in Figure 1c, TNF- tion of the production of TNF-induced pro-inflammatory stimulation of L929 cells also resulted in the rapid synthesis of factors. Therefore it is unclear whether necroptosis sup- an array of cytokines and chemokines, including: MCP-1, KC/ presses or enhances TNF-induced inflammatory responses. CXCL1, MIP-2, GMCSF, and IL-6. Similarly, primary murine Here we examine the inflammatory outcome of TNF- and embryonic fibroblasts (MEFs) also responded to TNFR lipopolysaccharide (LPS)-induced necroptosis and report that stimulation by synthesizing potent pro-inflammatory factors this mode of cell death resulted in a dramatic suppression of in the absence of any appreciable cell death (Supplementary the production of multiple TNF-induced cytokines and Figure S1a). chemokines, which was not compensated by the release of intracellular DAMPs. Knockdown of receptor-interacting pro- TNF induces RIPK3-dependent necroptosis in L929 cells tein kinase 3 (RIPK3) inhibited TNF-induced necroptosis and and MEFs. As has been demonstrated by many groups, restored pro-inflammatory cytokine production. Thus intracel- TNFR stimulation in RIPK3-epressing cells can be switched 3,30 lular pathogens encoding caspase inhibitors that skew TNF or to necroptosis through inhibition of caspase activity. As LPS signaling towards necroptosis may do so as an adaptive can be seen from Figures 1d–f and Supplementary Figure strategy to suppress the host inflammatory response. S1b, the poly-caspase inhibitor zVAD-fmk resulted in rapid TNF-induced necroptosis of L929 cells within 5–6h of receptor stimulation. TNF/zVAD-induced necroptosis Results was inhibited by the addition of Necrostatin-1 (Figure 1g) or through knockdown of RIPK3 (Figure 1h). Similarly, TNF induces the production of multiple pro-inflammatory MEFs also underwent necroptosis in response to TNF/zVAD cytokines and chemokines. Although TNF is often treatment (Supplementary Figure S1c), which was also considered a pro-apoptotic factor, this cytokine is predominantly RIPK3-dependent and inhibited by necrostatin (Supplementary an initiator of inflammation that is capable of transcriptionally Figures S1c and d). upregulating a battery of inflammatory gene expression events, in the absence of cell death. Indeed, the majority of TNF-induced necroptosis inhibits the production of cell types typically do not undergo apoptosis in response to cytokines and chemokines. We next examined the impact TNF unless transcription or translation is inhibited.13,14 To of TNF-induced necroptosis on the release of TNF-induced illustrate this, we conducted gene expression array analyses pro-inflammatory mediators. As can be seen from Figure 2a, on HeLa cells, which revealed dramatic upregulation of whereas TNF treatment of L929 cells resulted in a dose- numerous pro-inflammatory cytokines and chemokines in dependent production of high concentrations of cytokines response to TNF (Figure 1a), in the complete absence of cell and chemokines over 24 h, addition of zVAD-fmk dramatically death (Figure 1b). Thus TNF is highly pro-inflammatory suppressed the production of MCP-1, KC, MIP-2, RANTES whether cells die or not. and GMCSF. Importantly, concentrations of zVAD that did not Murine L929 cells have been widely used as a model efficiently evoke necroptosis did not result in cytokine system to explore the mechanism of TNF-induced pro- suppression (Figure 2b and Supplementary Figure S2a). grammed necrosis (i.e., TNF-induced cell death that occurs Thus the production of multiple TNF-induced cytokines and where caspases are inhibited). However, the impact of chemokines was suppressed as a consequence of triggering necroptosis on TNF-induced inflammatory events has not necroptosis. been explored to date. This is surprising, because TNF- A time course analysis of cytokine production in response to induced necroptosis is frequently considered to be more TNF or TNF/zVAD-fmk again revealed a dramatic suppression

Figure 1 TNF induces the secretion of multiple pro-inflammatory mediators irrespective of cell death. (a) HeLa cells were treated with TNF (10 ng/ml). After 6 h, cells were harvested and subjected to full genome microarray analysis. (b) HeLa cells were stimulated with TNF at the indicated concentrations. After 24 h, apoptosis was scored by morphology, and the cytokine/chemokine concentrations in the culture supernatants were determined by ELISA. (c) L929 cells were stimulated with TNF at the indicated concentrations. After 8 h, cell death was scored by morphology, and cytokine concentrations in the culture supernatants were determined by ELISA. (d) L929 cells were pretreated for 1 h in the presence or absence of zVAD (10 μM) followed by stimulation with TNF (10 ng/ml). After 8 h, cells were visualized by phase-contrast microscopy or H&E staining. (e) L929 cells were pretreated for 1 h in the presence or absence of zVAD (10 μM) followed by stimulation with TNF (10 ng/ml). At the indicated time points, cell death was analysed by AnnexinV/PI staining. (f) L929 cells were pretreated for 1 h in the presence or absence of zVAD (10 μM) followed by stimulation with TNF (10 ng/ml). After 24 h, cell death was analysed by PI staining. (g) L929 cells were pretreated for 1 h with the indicated concentrations of Nec1 and zVAD (10 μM), followed by addition of TNF (10 ng/ml). After 24 h, cell death was scored by morphology. (h) L929 cells were electroporated with either non-silencing siRNA or siRNA directed against RIPK1, RIPK3 or RIPK1 and RIPK3, as indicated. Forty-eight hours later, cells were pretreated in the presence or absence of zVAD (20 μM) for 1 h, followed by addition of TNF (10 ng/ml). After 5 h, cell death was scored by morphological assessment, and cell lysates were analysed by immunoblotting for levels of endogenous proteins. Error bars represent the mean ± S.E.M. of triplicate determinations from a representative experiment. For cell death analyses, triplicate counts were performed on a minimum of 300 cells per treatment. All data are representative of at least three independent experiments

Cell Death and Differentiation Necroptosis suppresses inﬂammation CJ Kearney et al 1315

HeLa Untreated -2 >10 TNF 100 1.2

Fold Gene Cell Death IL-8 %

IL-8 ( 80 1.0

100 Lymphotoxin

l i

CCL20 s 0.8

60 m /

50 CXCL1 o

t g

CCL2 p 0.6 n

CXCL10 o 40 CXCL3 p) 0.4 20 TNF As 20 CXCL2 0.2 CXCL11 10 IL-6 0 10 5 1 0.5 S100A3 0 10 5 1 0.5 IL7R SAA2 TNF (ng/ml) TNF (ng/ml) 5 ICAM4 ICAM1 8 250 SAA1 1.2 CXCL1 IL-6 iCAM RANTES IL-1β 7 1.0 IL-32 1.0 200

6 0.8 l

AREG l

l l 5 m RANTES 0.8 /

3 m 150

m /

0.6 g

CCL19 / g

g 4 p

IL-11 0.6 g

n n n 3 0.4 100 CCL28 0.4 IL-15 2 1 CDNF 0.2 0.2 50 LDB2 1 S100Z ATXN3 0 10 5 SGSM1 0 10 5 1 0.5 0 10 5 1 0.5 1 0.5 0 10 5 1 0.5 ZNF618 TNF (ng /ml) TNF (ng/ml) TNF (ng/ml) TNF (ng/ml)

L929 Untreated TNF TNF+ZVAD Untreated TNF 8 hours TNF 24 hours

Phase

100 50 Cell Death MCP-1 40 KC

) 80 40 H&E %

( 30

l l

i 60 30

g c

n 20 n

e 40 20 N 20 10 10 L929 100 Necrosis 0 20 10 5 1 0.5 0 20 10 5 1 0.5 0 20 10 510.5 80 Untreated TNF (ng/ml) TNF (ng/ml) TNF (ng/ml) TNF 150 60 TNF+zVAD 0.8 MIP-2 GMCSF 250 IL-6

200 40

l l

0.6 100 l

/ /

/ 150

AnnexinV / PI (%) PI / AnnexinV

n p 0.4 p 20 50 100 0.2 50 2 4 6 8 10 20 0 20 10 5 1 0.5 0 20 10 5 1 0.5 0 20 10 5 1 0.5 Hours TNF (ng/ml) TNF (ng/ml) TNF (ng/ml)

siRNA: NS NS RIPK1RIPK3 RIPK1+3 83 RIPK1 62 RIPK3 Untreated zVAD Untreated TNF + zVAD 47 100 Actin Necrosis Cell Death 100 Cell Death 100 80 80 80 NS oligo 60 60 NS oligo 60 RIPK1 40 40 RIPK3 Necrosis (%) Necrosis 40

PI Uptake (%) Uptake PI 20 RIPK1+3 20 (%) Necrosis 20 0 20 10 5 1 0.5 0 40 20 10 5 2.5 Unt TNF TNF+ TNF (ng/ml) Nec1 (μM) zVAD

Cell Death and Differentiation Necroptosis suppresses inﬂammation CJ Kearney et al 1316

L929 100 Necrosis MCP-1 Untreated TNF Untreated TNF

) 100 80 100

% l ( 80 Necrosis MCP-1

m 25

h /

t 60 g

) a

n 80

e 60

(

D 40 20 l

e l 40 l

k e

a 60 m

t /

C 20

p 20 g 15

P 40 zVAD - + - + - ++- - + - + zVAD - + - + - + - + - ++- 10 TNF (ng/ml) TNF (ng/ml) 20 5 2.5 MIP-2 200 RANTES 2.0 0 20 10 51 0 20 10 5 1

l 150

m /

1.5 / zVAD (μM) zVAD (μM)

g n

pl 100 1.0 50 Untreated TNF Untreated TNF 0.5 16 KC MIP-2 zVAD - ++- - ++- - ++- zVAD - + - ++- - + - + - + 14 2.0 TNF (ng/ml) TNF (ng/ml) 12

70 1.0 l 10 1.5

m KC GMCSF m

/ 60 /

n 8 0.8 nl

l 50 1.0

m /

m 6 /

40 g 0.6

g nl n 4 30 0.4 0.5 20 2 10 0.2 0 20 10 5 1 0 20 10 51 zVAD - ++++- - - - + - + zVAD - + - ++- - + - + - + zVAD (μM) zVAD (μM) TNF(ng/ml) TNF (ng/ml)

Necrosis 64 100 MCP-1

) 56 %

( 80 48 h

t Untreated

m a

60 / 40 e

g TNF nl

D 32 l l 40 TNF+zVAD

e 24

C 16 20 8

2 4 6 8 10 12 14 16 18 20 22 24 246810 12 14 16 18 20 22 24 hours hours 35 KC 800 MIP-2 30

25 l

600 Untreated

m /

/ 20 g

g TNF

p nl 15 400 TNF+zVAD 10 5 200

2 468101214 16 18 20 22 24 246810 12 14 16 18 20 22 24 hours hours MEFs 100 16 Necrosis 140

80 14 KC MCP-1 %

( 12 120

l t

60 10 l 100

m /

g 8 80

g n

l 40 n

l 6 60 e

C) 20 4 40 2 20

zVAD - + - + - + - + - + zVAD - + - + - + - + - + zVAD - + - + - + - + - + TNF (ng/ml) TNF (ng/ml) TNF (ng/ml) 20 1.0 RANTES 500 IL-6 GMCSF 0.8

15 400

/ m

m 0.6

/ /

g 300

g g

nl 10

n pl 200 0.4 5 100 0.2

zVAD - + - + - + -++ - zVAD - + - + - + - + - + zVAD - + -++ -+- - + TNF (ng/ml) TNF (ng/ml) TNF (ng/ml)

Cell Death and Differentiation Necroptosis suppresses inﬂammation CJ Kearney et al 1317 of cytokine production during necroptosis, which correlated caspase inhibition, interfered with RIPK1-dependent recruit- with much earlier cell death under necroptotic conditions ment of NEMO/IKKγ and NFκB activation downstream. (Figure 2c). We also observed a similar dramatic reduction in Indeed, Dixit and colleagues have previously reported that TNF-induced pro-inflammatory cytokine production upon RIPK3 suppresses RIPK1-dependent NFκB activation upon inducing necroptosis in MEFs (Figure 2d). transient overexpression.31 Thus it was formally possible that Some cells types are resistant to necroptosis under other effects of caspase inhibition (such as stabilization of the conditions of caspase inhibition alone but can be sensitized deubiquitinating enzyme cylindromatosis), as opposed to by neutralizing IAPs or inhibiting transcription/translation in RIPK3-dependent cell death, were responsible for suppres- tandem with caspase inhibition (Supplementary Figures S2b sing TNF-induced inflammatory cytokine production. and c). In this context, we also found that inducing necroptosis To explore whether RIPK3-dependent cell death was the of HT29 cells or MEFs using TNF/zVAD in the presence of IAP primary reason for the suppression of TNF-induced cytokine antagonist also dramatically suppressed cytokine production production under necroptosis-inducing conditions, we (Supplementary Figures S2b and c). silenced RIPK3 expression, followed by stimulation with TNF/zVAD to assess the effects on TNF-induced necroptosis Caspase activity is not required for TNF-induced and cytokine production. As shown in Figure 4a, knockdown of cytokine and chemokine synthesis. Although we only RIPK3 blocked TNF-induced necroptosis and also partly observed suppression of TNF-induced cytokine production restored TNF-induced cytokine production in the presence of when necroptosis occurred, it was formally possible that zVAD zVAD. Similarly, knockdown of the downstream target of was having a direct inhibitory affect on TNF-induced cytokine RIPK3, MLKL, also blocked TNF/zVAD-induced necroptosis production. To exclude the possibility that zVAD had a direct and restored cytokine production, suggesting that termination inhibitory effect on TNF-induced cytokine production, without of cell viability via RIPK3 and MLKL activation was responsible recourse to necroptosis, we used HeLa cells as these cells for the decline in cytokine synthesis. lack RIPK3 and thus fail to undergo necroptosis in response to We also examined cytokine and chemokine production TNF/zVAD treatment. Treatment with zVAD efficiently blocked within 5 h of stimulation of TNF alone, or TNF/zVAD, to ask TRAIL-induced apoptosis in HeLa cells (Figure 3a) but failed to whether cytokine production or NFκB activation upstream cause necroptosis when combined with TNF (Figure 3b). were attenuated prior to significant cell death (Figures 4b Under these conditions, zVAD failed to suppress TNF-induced and c). These experiments suggested that early TNF-induced cytokine production (Figures 3c–f and Supplementary Figures cytokine production (Figure 4b), as well as NFκB and MAPK S3a and b), indicating that necroptosis is responsible for the activation (Figure 4c), were highly similar in response to TNF suppression of cytokine production in RIPK3-expressing cells and TNF/zVAD treatment, again suggesting that the decline in treated with TNF/zVAD. Furthermore, zVAD also potently cytokine synthesis seen under necroptosis-inducing condi- inhibited Fas-induced apoptosis in HeLa cells (Supplementary tions was due to cell death rather than impaired signal Figure S3c), and consistent with this, Fas-induced IL-6 and KC transduction. were increased, rather than suppressed, in the presence of Because the RIPK1 inhibitor, necrostatin-1, can block TNF/ zVAD due to restoration of cell viability (Supplementary zVAD-induced necroptosis, we also used this approach to ask Figures S3d and e). Furthermore, zVAD failed to exert a whether blocking cell death under necroptotic-inducing con- suppressive affect on TNF-induced MCP-1 and KC in ditions restored cytokine production. Necrostatin-1 efficiently murine 3LL cells, which are also refractory to necroptosis blocked TNF/zVAD-induced necroptosis, as expected (Figures 3g–i). We also confirmed that zVAD did not inhibit (Figure 4d). However, we also observed that necrostatin had TNF-induced cytokine synthesis at the mRNA level, in the robust inhibitory effects on TNF-induced KC and MIP-2 absence of cell death (Figures 3j and k). production, in the absence of zVAD (Figure 4d). This suggests that at least some of the anti-inflammatory effects that have Knockdown of RIPK3 or MLKL restores TNF-induced been attributed to necrostatin-1 in previous studies could be cytokine production. As noted above, a straightforward due to direct effects on TNF-induced cytokine production, reason for necroptosis-mediated suppression of TNF-induced rather than due to inhibition of necroptosis. However, this cytokine production was simply because cells underwent aside, we also observed that necrostatin treatment did restore rapid cell lysis, thereby terminating cytokine production. MCP-1 and KC production in response to TNF/zVAD treat- However, an alternative reason could be that excessive ment to some degree (Figure 4d). To investigate the effects of recruitment of RIPK3 onto RIPK1, as a consequence of necrostatin-1 on TNF-induced cytokines further, we also used

Figure 2 Necroptosis suppresses TNF-induced cytokine and chemokine production. (a) L929 cells were pretreated for 1 h in the presence or absence of zVAD (20 μM), followed by addition of TNF to a final concentration of 20, 10, 5, 1 or 0.5 ng/ml (from left to right). After 24 h, cell death was scored by morphology, and cytokine/chemokine concentrations in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). (b) L929 cells were pretreated for 1 h with the indicated concentrations of zVAD, followed by addition of TNF (10 ng/ml). After 24 h, cell death was analysed by PI staining, and the culture supernatants were harvested to measure chemokines by ELISA. (c) L929 cells were pretreated for 1 h with zVAD (20 μM), followed by addition of TNF (10 ng/ml). At the indicated time points, cell death was scored by morphology, and the culture supernatants were harvested to measure chemokines by ELISA. (d) MEFs were pretreated for 1 h with zVAD (25 μM), followed by addition of TNF to 100, 50, 25 or 12 ng/ml (from left to right). After 24 h, cell death was scored by morphology, and cytokine/chemokine concentrations in the culture supernatants were determined by ELISA. Error bars represent the mean ± S.E.M. of triplicate determinations from a representative experiment. For cell death analyses, triplicate counts were performed on a minimum of 300 cells per treatment. All data are representative of at least three independent experiments

Cell Death and Differentiation Necroptosis suppresses inﬂammation CJ Kearney et al 1318

HeLa Untreated TRAIL Untreated zVAD Untreated zVAD 40 100 800 Cell Death Cell Death 700 IL-8

)

% 80 600 30 (

e / 500

k 60 g

pl 20 t 400

u 40 300

10 P 200

20 100 AnV/PI positive (%) positive AnV/PI

010510.5 0 5 2.5 10.5 0.1 0 5 2.5 10.5 0.1 zVAD (μM) TNF (ng/ml) TNF (ng/ml)

Untreated zVAD Untreated zVAD Untreated zV AD 2.5 6 2.5 CXCL1/KC IL-6 RANTES 2.0 5 2.0

l l 4 l

m 1.5 m m / / / 1.5 g g 3 g n 1.0 n n 2 1.0 0.5 1 0.5 0 5 2.5 10.5 0.1 0 5 2.5 1 0.10.5 0 52.510.5 0.1 TNF (ng/ml) TNF (ng/ml) TNF (ng/ml)

Mouse 3LL Untreated TNF Untreated TNF Untreated TNF 100 5 8 Cell Death MCP-1 7 KC 80 4 6

l l 5

m 60 / 3

g 4

n 40 2 n 3 PI uptake(%) 2 20 1 1 0201052.5 0201052.5 0201052.5 zVAD (μM) zVAD (μM) zVAD (μM)

HeLa mRNA mRNA mRNA mRNA

4.0 MCP-1 ns n 15 CXCL1 ns MCP-1

n n RANTES

o o 15 s ns

i 3.6 (KC) o

2.2 i

3.2 s

r r 10

2.8 x

p p 1.8

p 10

E 2.4 x

d d 2.0

l 1.4 d 5 l

1.6 o 5

F 1.2 1.0 F

TNF TNF TNF Hours: 02460246

Untreated Untreated Untreated TNF TNF/zVAD TNF+zVAD TNF+zVAD TNF+zVAD mRNA mRNA mRNA mRNA 5.0 IL-6 3.5 ns ns ns n 80 GM n IL-6 TNF o

n 15

i o 4.5

s o 70

s i CSF

3.0 s

s s 4.0

s 60

e e 3.5

r 2.5 50 p 10

x p 3.0

40 E

E 2.0 2.5 d

30 l

l o 2.0 5

o o 1.5 20 F

F F 10 1.5 1.0 Hours: TNF TNF TNF 02460246 TNF TNF/zVAD Untreated Untreated Untreated TNF+zVAD TNF+zVAD TNF+zVAD

Cell Death and Differentiation Necroptosis suppresses inﬂammation CJ Kearney et al 1319

MEFs, which do not die in response to TNF alone necroptosis enhances or suppresses TNF-induced inflam- (Supplementary Figure S2). Once again, we observed that mation depends on the relative potency of endogenous necrostatin-1 significantly inhibited secretion of KC, RANTES DAMPs, which are liberated via necroptosis, versus TNF- and IL-6 in the absence of cell death or caspase inhibition induced pro-inflammatory cytokines and chemokines in (Supplementary Figure S4). driving inflammatory processes in vivo. To explore this issue, we introduced supernatants from L929 Necroptosis suppresses LPS/TLR4-induced cytokine cells treated with TNF alone or TNF/zVAD (necroptotic) into production. Because necroptosis can also be invoked in the peritoneal cavities of immunocompetent BALB/c mice and response to LPS stimulation, we next explored whether this monitored the peritoneal exudates after 24 h to explore also blunted pro-inflammatory cytokine and chemokine whether supernatants from necroptotic cells were more or production in this context. As shown in Figure 5a, LPS less inflammatory than those from cells treated with TNF induced robust necrosis in primary bone marrow-derived alone. As shown earlier, supernatants from TNF-treated cells macrophages (BMDMs) in the presence of zVADfmk. LPS/ contained much higher concentrations of pro-inflammatory zVAD-induced necroptosis was partly inhibited by addition of cytokines than those from cells treated with TNF/zVAD necrostatin-1 (Figure 5b) or through knockdown of RIPK3 (Figure 6a). Consistent with this, supernatants from TNF- (Figure 5c). However, a component of the LPS-induced treated cells induced robust infiltration of cells into the necrosis observed was not necrostatin-1-inhibitable or peritoneal cavities of mice (Figure 6b), with increases in the blocked by RIPK3 knockdown. Moreover, similar to what we percentages of neutrophils, lymphocytes, monocytes and observed with TNF, LPS-induced IL-6, MIP-2, RANTES, KC small peritoneal macrophages (Figures 6c and d). In contrast, and MCP-1 production were also considerably blunted under supernatants from TNF/zVAD-treated (necroptotic) cells necroptosis-inducing conditions (Figure 5d). exhibited little increase in peritoneal cellularity (Figure 6b), or It is important to note that IL-1β, a key LPS-induced cytokine, increases in neutrophils, lymphocytes or monocytes, consis- requires caspase-1 activity,which is blocked under necroptosis- tent with our in vitro observations. Thus necroptosis attenu- inducing conditions. Thus LPS/zVAD treatment greatly reduced ates rather than exacerbates TNF-induced inflammation. the production of mature IL-1β as expected (Figure 5e). To explore which chemokines were having the dominant Consistent with this, supernatants from LPS/zVAD-treated role in recruiting cells to the peritoneum in response to TNF BMDMs had greatly reduced pro-inflammatory activity when treatment (Figure 6b), we also performed chemotaxis assays transferred onto HeLa cells (as measured by the production of ex vivo using peritoneal exudate cells from TNF-treated mice. IL-8 from the latter), when compared with supernatants from Chemotaxis of peritoneal exudate cells was measured in BMDMs treated with LPS alone (Figure 5f). Using MEFs, which response to supernatants from TNF-treated cells that were do not engage RIPK3 or undergo necroptosis upon LPS/zVAD either mock depleted (IgG) or depleted with anti-MCP-1, anti- treatment, we also confirmed that zVAD did not suppress LPS- KC or anti-MIP-2 monoclonal antibodies (Figure 6e). As induced chemokines independently of necroptosis (Figure 5g). shown in Figure 6f, chemotaxis of peritoneal exudate cells was Similar results were also observed using THP-1 cells, which largely abolished upon depletion of MCP-1 from the super- failed to under necroptosis in response to LPS/zVAD treatment natants, consistent with the very high concentrations of this (Supplementary Figure S5b). chemokine produced in response to TNF (Figure 6a). Collectively, the above data indicate that necroptosis attenuates the production of numerous LPS-induced pro- LPS-induced inflammation is suppressed through inflammatory cytokines in RIPK3-expressing cells via termina- caspase inhibition. As demonstrated earlier, LPS also tion of cell viability and through inhibition of caspase activity, promotes necroptosis in the presence of caspase inhibition which is required for IL-1β maturation. (Figure 5a), which led to suppression of the production of LPS-induced cytokines in vitro (Figures 5d and e). To explore TNF-induced necroptosis results in reduced inflamma- whether caspase inhibition also attenuated LPS-driven tion in vivo. The preceding experiments demonstrated that inflammation in vivo, we treated balb/c mice with LPS TNF or LPS-induced cytokine and chemokine production intraperitoneally in the presenece or absence of zVAD-fmk, was dramatically suppressed under necroptosis-inducing to promote necroptosis. Previous studies have shown that conditions. However, a caveat is that necroptosis liberates LPS induces cell death in the peritoneal cavity, as well intracellular constituents, some of which can function as as activation and efflux of resident macrophages and DAMPs, that could potentially compensate for the loss lymphocytes.32,33 The latter events are mirrored by an of cytokine and chemokine production. Thus whether increase in splenic cellularity, composed of migrating

Figure 3 Caspase activity is not required for TNF-induced cytokine production. (a) HeLa cells were pretreated for 1 h with the indicated concentrations of zVAD, followed by addition of TNF-related apoptosis-inducing ligand (TRAIL; 100 ng/ml). After 8 h, cell death was analysed by AnnexinV/PI staining. (b–f) HeLa cells were pretreated for 1 h in the presence or absence of zVAD (10 μM), followed by addition of TNF (10 ng/ml). After 24 h, cell death was analysed by AnnexinV/PI staining, and cytokine/chemokine concentrations in the culture supernatants were determined by ELISA. (g–i) Murine 3LL cells were pretreated for 1 h with the indicated concentrations of zVAD, followed by addition of TNF (10 ng/ml). After 24 h, cell death was analysed by PI staining, and chemokine concentrations in the culture supernatants were determined by ELISA. Error bars represent the mean ± S.E.M. of triplicate cell cultures. (j) HeLa cells were seeded at 5 × 105 in 6-cm dishes. The following day, cells were pretreated for 1 h with zVAD (10 μM), followed by addition of TNF (10 ng/ml). After 4 h, or at the indicated time points (k), total RNA was isolated, reverse transcribed and used to seed real-time PCR reactions. Error bars represent the mean ± S.E.M. of triplicate cell cultures

Cell Death and Differentiation Necroptosis suppresses inﬂammation CJ Kearney et al 1320

TNF TNF+zVAD 100 35 NS oligo Necrosis MCP-1

NS NS RIPK3RIPK3 (1) MLKL (2) MLKL (1) (2) NS oligo ) 80 30 % RIPK3 (1) ( 25

RIPK3 l s

RIPK3 (2) i

60 m s / 20

MLKL (1) o

Actin g

r n

MLKL (2) c 40 15 e

N 10 100 Necrosis MCP-1 20 *** 5 30 80 *** *** 0.25 1 2 3 4 5 0.25 1 2 3 4 5 60 20 hours hours 70

40 ng/ml KC 700 IL-6 10 60 600 Cell Death (%) 20

50 500

m /

m 40

/ 400

g g Buffer TNF TNF/zVAD Buffer TNF TNF/zVAD 30 pl n 300 20 200 MIP-2 KC *** 0.8 40 10 100 0.25 1 2 3 4 5 0.25 1 2 3 4 5 30 0.6 *** hours hours

ng/ml ng/ml 0.4 1.4 20 MIP-2 RANTES 1.2 80 10 0.2 1.0 60

0.8

/ g

Buffer TNF TNF/zVAD Buffer TNF TNF/zVAD 0.6 g 40

nl pl 0.4 Untreated TNF TNF+zVAD 20 0.2 Mins: 0 15 30 60 90 0 15 30 60 90 0 15 30 60 90 IKBα 0.25 1 2 3 4 5 32 0.25 1 2 3 4 5 hours hours 62 P p65 100 p65 62 100 Necrosis MCP-1 p44 80

P ERK ) 80 47 p42 % 47 ( 60

p44 h

t ERK 60 m

/ p42 a

nl D 40 40 l

P p38 l

47 e C 20 20 47 p38

47 Actin Buffer Nec zVAD zVAD Buffer Nec zVAD zVAD Nec Nec TNF TNF TNF TNF TNF TNF TNF TNF+zVAD TNF TNF hours: 0 0.25 1 2 3 4 5 0 0.25 1 2 3 4 5

32 IKBα 250 KC MIP-2 0.6 62 P p65 200

l 62 p65 l 150 0.4

g 47 p44 g

n P ERK n p42 100 47 p44 0.2 ERK p42 50

32 P p38 Buffer Nec zVAD zVAD Buffer Nec zVAD zVAD p38 Nec Nec 32 TNF TNF TNF TNF TNF TNF TNF TNF 47 Actin

Cell Death and Differentiation Necroptosis suppresses inﬂammation CJ Kearney et al 1321 neutrophils, macrophages, monocytes and clonally expand- participation. The necroptosis effectors RIPK3 and MLKL, in ing lymphocytes.33,34 addition to being able to promote necroptosis under conditions As shown in Figure 7a, LPS administration led to a dramatic where caspase activity is blocked, are very likely to have other reduction in peritoneal cellularity, which was significantly cellular functions, which remain unknown at present. However, inhibited in LPS/zVAD-treated animals. However, it should we propose that these molecules could also be regarded as also be noted that zVAD alone also led to a reduction in negative regulators of TNF- and LPS-induced inflammation, peritoneal cellularity. Consistent with this, we observed an as their activation rapidly shuts down cytokine synthesis increase in splenic cellularity in response to LPS, which was through cell lysis. From this perspective, our findings also have attenuated in the presence of zVAD Figure 7b. We also major implications for the interpretation of the pathology of confirmed that LPS/zVAD treatment caused a significant RIPK3 null animals in response to pathogen challenge as well increase in peritoneal cell death, compared with LPS alone as sterile injury. RIPK3 null animals are often protected from Figure 7c. pathogen challenge or injury-induced inflammation, and this is As shown in Figure 7d, LPS treatment induced robust frequently attributed to blocking necroptosis. However, our increases in splenic neutrophils, F4/80+ macrophages, data suggest that RIPK3 null animals would make more eosinophils and B- and T-lymphocytes, which were all effective and prolonged immune responses through prevent- diminished in the presence of LPS/zVAD. This is consistent ing the shutdown of cytokine/chemokine synthesis that would with a suppression rather than enhancement of LPS-induced otherwise occur via necroptosis. Viewed in this light, our data inflammatory responses under necroptotic (caspase inhibi- also cast doubt upon the view that necroptosis is invariably a tion) conditions. Once again, these data argue that necro- host response to pathogens encoding caspase inhibitory ptosis suppresses rather than enhances inflammatory proteins. Instead, it is possible that necroptosis could also responses, most likely through killing responding cells and serve as a pathogen-driven mechanism to limit the host reducing inflammatory cytokine production, as well as through inflammatory response in at least some contexts. Thus blunting the production of IL-1β and IL-18 in a caspase- infectious agents that promote necroptosis may do so as a dependent manner. mechanism to neutralize host immune responses by rapidly To explore this further, we recovered peritoneal cells from terminating conventional cytokine and chemokine production. PBS-treated mice and stimulated with either LPS or LPS/ In this situation, the liberation of endogenous DAMPs as a zVAD to evaluate cytokine production ex vivo. LPS/zVAD- consequence of necroptosis may be insufficient to compen- treated peritoneal exudates exhibited increases in cell death sate for the loss of cytokine and chemokine synthesis. as compared with cells treated with LPS or zVAD alone Support for our observations come from a study by (Figure 7e). Furthermore, we again observed dramatic Linkermann et al.35 who reported that the necroptosis inhibitor, reductions in LPS-induced TNF, MCP-1, KC, MIP-2 and necrostatin-1, failed to protect from TNF/zVAD-induced shock RANTES in the LPS/zVAD (necroptotic) condition (Figure 7e). and lethality but rather accelerated time to death compared Taken together, the above data argue that TNF- or LPS- with TNF/zVAD treatment alone. This is consistent with our induced necroptosis is significantly less pro-inflammatory than observations that necroptosis suppresses the inflammatory unimpeded TNF or LPS stimulation. Our data also argue that effects of TNF, which can lead to lethality, rather than conventional TNF- or LPS-induced cytokines and chemokines enhancing these effects as is frequently speculated. Suppres- exert more potent pro-inflammatory effects than endogenous sing necroptosis in the context of TNF stimulation is likely to DAMPs that are released during necrosis or necroptosis. enhance the lethal effects of this cytokine through prolonging and exacerbating the cytokine storm. Moreover, Linkerman et al.35 also observed similar effects with cerulein-induced Discussion pancreatitis, where necrostatin-1 worsened the inflammatory Here we have shown that, in contrast to current interpretations symptoms, again most likely by restoring production of a of the biological consequences of necroptosis, RIPK3- plethora of TNF-inducible inflammatory mediators. However, it dependent cell death resulted in a dampening of TNF- should also be noted that interpreting effects seen with induced pro-inflammatory cytokine/chemokine secretion, necrostatin-1 in vivo is very problematic, as necrostatin may thereby leading to a less inflammatory outcome. Similarly, have direct inhibitory effects on the production of some TNF- we also found that LPS-initiated necroptosis was less induced cytokines as we have shown (Figure 4 and inflammatory than LPS stimulation involving caspase Supplementary Figure S4). In particular, we have found that

Figure 4 RIPK3- and MLKL-dependant termination of cell viability attenuates TNF-induced cytokine and chemokine production. (a) L929 cells were electroporated with either non-silencing siRNA or siRNA directed against RIPK3 and MLKL, as indicated. Forty-eight hours later, cells were pretreated in the presence or absence of zVAD (10 μM) for 1 h, followed by addition of TNF (10 ng/ml). After 24 h, cell death was scored by morphology, and cytokine/chemokine concentrations in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). (b) L929 cells were pretreated for 1 h in the presence or absence of zVAD (10 μM), followed by addition of TNF (1 ng/ml). At the indicated time points, cell death was scored by morphology, and cytokine/chemokine concentrations in the culture supernatants were determined by ELISA. (c) L929 cells were pretreated for 1 h in the presence or absence of zVAD (10 μM), followed by addition of TNF (10 ng/ml). Cell lysates were prepared at the indicated time points and analysed by immunoblotting for levels of endogenous proteins. (d) L929 cells were pretreated in the presence or absence of Nec1 (5 μM) and/or zVAD (10 μM), followed by addition of TNF to 20, 10, 5 or 2.5 ng/ml (from left to right). After 24 h, cell death was scored by morphology, and cytokine/chemokine concentrations in the culture supernatants were determined by ELISA. Error bars represent the mean ± S.E.M. of triplicate determinations from a representative experiment. For cell death analyses, triplicate counts were performed on a minimum of 300 cells per treatment. All data are representative of at least three independent experiments

Cell Death and Differentiation Necroptosis suppresses inﬂammation CJ Kearney et al 1322

BMDM siRNA: NS NS RIPK1RIPK3 RIPK1+3 Untreated zVAD 83 100 80 RIPK1 Cell Death Cell Death 62 RIPK3 80 60 47 Actin 60 Untreated 40 LPS Cell Death 40 LPS+zVAD 80

) PI Uptake (%) NS oligo

PI Uptake (%)

% ( 60 NS oligo

20 e

20 k RIPK1

a t RIPK3 p 40

U RIPK1+3

0 1.0 0.5 0.10.25 2 0 40 20 10 5 2.5 P 20 LPS (μ g/ml) Nec1 (μM) Unt LPSLPS+ zVAD BMDM Untreated LPS zVAD LPS+zVAD 104 104 4 104 13 4 43 13 10 31 6 55 37 3 3 3 10 10 103 10

2 2 2

PI PI PI PI 10 10 102 10 1 1 44 1 10 82 10 101 10 61 9 101 102 103 104 101 102 103 104 101 102 103 104 101 102 103 104 AnnexinV AnnexinV AnnexinV AnnexinV 160 20 120 RANTES KC MCP-1 TNF 800 140 100 120 15 80 600 100 80 10 60 400 ng/ml pg/ml ng/ml 60 ng/ml 40 200 40 5 20 20

zVAD - + - + - + - + - + zVAD - + - + - + - + - + zVAD - + - + - + - + - + zVAD - + - + - + - + - + LPS LPS LPS LPS

BMDM Inﬂammasome HeLa Bioassay 300 100 IL-6 MIP-2 300 IL1-β 160 IL-8 250 80 250 200 120 60 200

ng/ml 150 ng/ml pg/ml pg/ml 150 80 40 100 100 20 40 50 50

zVAD - + - + - + - + - + zVAD - + - + - + - + - + zVAD - + - + - + - + - + zVAD - + - + - + - + - +

LPS LPS LPS LPS

MEFs Untreated LPS Untreated LPS Untreated LPS Untreated LPS 100 20 1.4 Necrosis 12 MCP-1 KC RANTES 1.2 80 10 15 1.0 60 8 0.8 10

ng/ml 6 ng/ml ng/ml 40 0.6

PI Uptake (%) 4 0.4 20 5 2 0.2

0201052.5 0201052.5 0201052.5 0201052.5 zVAD (μM) zVAD (μM) zVAD (μM) zVAD (μM)

Cell Death and Differentiation Necroptosis suppresses inﬂammation CJ Kearney et al 1323

TNF-induced production of IL-6, which is a key player in response, rather than a host response designed to terminate models of severe systemic inflammation (SIRS) induced by viral replication. TNF, is dramatically reduced in the presence of necrostatin Infection with the intracellular bacterium Salmonella typhi- (Supplementary Figure S4). Indeed, direct effects of murium has also been shown to lead to RIPK3-dependent necrostatin-1 on TNF-induced cytokine production might well necroptosis.38 However, infection of RIPK3 null macrophages explain the protection afforded by this kinase inhibitor during (incapable of undergoing necroptosis) led to decreased TNF-induced shock in vivo,27 especially as the latter effects bacterial burdens, suggesting that rapid RIPK3-dependent were seen in the absence of caspase inhibition where necroptosis of macrophages is beneficial to the pathogen and necroptosis would not be expected. helps to attenuate the host immune response. Whether necroptosis is beneficial to the host, or to Neutrophils are rapidly recruited from the peripheral pathogens, may be context-dependent. RIPK3 null mice circulation to sites of infection via chemokine gradients display increased susceptibility to replication of vaccinia generated by tissue-resident innate immune cells, where they virus.9 Thus, in this instance, necroptosis may be beneficial deploy aggressive anti-microbial mechanisms. Local macro- to the host by depriving the virus time to replicate. Further- phages, which provide a rich source of neutrophil chemoat- more, cytomegalovirus encodes caspase inhibitors and vIRA, tractants KC and MIP-2, are required for neutrophil migration which disrupts the host RHIM-dependent RIPK1 and RIPK3 into skin infected with Staphyloccus aureus and subsequent interaction to promote pathogenesis.10 However, the role of bacterial clearance.39 However, S. aureus utilizes virulence necroptosis as an anti-viral strategy appears to be more factor hemolysin to promote rapid macrophage necrosis at the complex and context dependent than initially presumed. infection site, thereby dampening chemokine generation and Previous studies using poxviruses that encode caspase neutrophil recruitment.39 This demonstrates the importance of inhibitors have shown that virus deficient in cytokine response the initial chemokine wave that is released in response to modifier A (CrmA), a potent Caspase-1 and -8 inhibitor, is less pathogen infection and highlights responder cell necrosis as a pathogenic than wild-type virus capable of inducing necrop- pathogen strategy to terminate it. Indeed, we also found that tosis and processing IL-1β.36 Although it is unclear whether synthesis of chemokines KC and MIP-2 was rapidly termi- CrmA evolved to suppress caspase-1-mediated IL-1β matura- nated as a consequence of TNF- and LPS-induced necrop- tion or to promote necroptosis through caspase-8 inhibition, it tosis (Figures 3b and 5d) and translated into impeded suggests that the ability to inhibit caspase activity, thus neutrophil recruitment to the peritoneal cavity (Figure 6c) promoting necroptosis-inducing conditions, can be beneficial and spleen (Figure 7d) in vivo. to the virus rather than the host in at least some situations. In conclusion, here we have provided evidence to Similarly, rabbits infected with myxoma virus lacking the CrmA argue that necroptosis may be less inflammatory than the homolog Serp2 display increased viral control.37 alternative outcomes (cell survival or apoptosis) due to rapid The preceding observations suggest that promoting necrop- cessation of the transcriptional pro-inflammatory programme tosis can be beneficial to viruses in certain contexts. This fits induced by TNF or LPS. Thus viruses and other microorgan- well with the implications of our observations and suggests isms that evoke this mode of cell death may well do so that some viruses may deliberately shut down the host as an adaptive strategy to minimize the host inflammatory inflammatory response by invoking necroptosis in a cell that response. receives TNF stimulation. This might also have the consequence of facilitating viral spread by killing the very cells that Materials and Methods act as sentinels in tissues vulnerable to viral infection. It is also Materials. The following antibodies were used: anti-RIPK1, anti-IκB (BD, relevant to note that were necroptosis always an effective Oxford, UK), RIPK3 (ProSci, Poway, CA, USA), anti-phospho-p65, anti-phospho host-response to viral infection, designed to terminate viral ERK, anti-ERK, anti-phospho-p38antip38 (Cell Signaling, Hitchin, UK), and anti p65 (Santa Cruz, Dallas, TX, USA). Recombinant TNFα was purchased from Roche replication by depriving the virus of a host, then it would be (Dublin, Ireland). zVAD-fmk was from Bachem (Bubendorf, Switzerland); Nec1 and expected that viruses would rapidly emerge that have lost their LPS were from Sigma (Arklow, Ireland). caspase inhibitory protein(s). However, the fact that multiple viruses do encode caspase inhibitors strongly suggests that Cell culture. HeLa and L929 cells were cultured in RPMI media (Gibco, these are beneficial to viral replication or spread. Thus viral Renfrew, UK), supplemented with 5% fetal calf serum (FCS). MEFs, BMDMs and initiation of necroptosis could also be viewed a strategy HT-29 cells were cultured in DMEM (Gibco) supplemented with 10% FCS. Cells adopted by certain viruses to suppress the host inflammatory were cultured at 37 °C in humidified atmosphere with 5% CO2.

Figure 5 TLR4-dependent necroptosis suppresses pro-inflammatory cytokine production. (a) BMDMs were pretreated for 1 h with zVAD (15 μM) or not, followed by addition of LPS to the indicated concentrations. Forty-eight hours later, cell death was scored by PI staining. (b) BMDMs were pretreated for 1 h with the indicated concentrations of Nec1 in the presence or absence of zVAD (15 μM), followed by addition of LPS (1 μg/ml). After 24 h, cell death was scored by PI staining. (c) BMDMs were electroporated with either non-silencing siRNA or siRNA directed against RIPK1, RIPK3 or RIPK1 and RIPK3, as indicated. Forty-eight hours later, cells were pretreated for 1 h in the presence or absence of zVAD (15 μM), followed by addition of LPS (1 μg/ml). After 24 h, cell death was scored by PI staining. (d) BMDMs were pretreated for 1 h in the presence or absence of zVAD (15 μM), followed by addition of LPS to 2, 1, 0.5 and 0.25 μg/ml (left to right). Forty-eight hours later, cell death was scored by PI staining, and cytokine/chemokine concentrations in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). (e) BMDMs were pretreated for 1 h in the presence or absence of zVAD (15 μM), followed by addition of LPS to the indicated concentrations. Forty-eight hours later, culture supernatants were analysed for mature IL-1β by ELISA. (f) HeLa cell cultures were treated with 100 μl of the BMDM supernatent from panel (e). After 24 h, HeLa supernatents were analysed for IL-8 by ELISA (HeLa bioassay). (g) MEFs were pretreated for 1 h with the indicated concentrations of zVAD, followed by addition of LPS (5 μg/ml). After 24 h, cell death was analysed by PI staining, and chemokine concentrations in the culture supernatants were determined by ELISA

Cell Death and Differentiation Necroptosis suppresses inﬂammation CJ Kearney et al 1324

Necrosis 4 Untreated TNF TNF+zVAD 100 10 4 104

10 )

80 %

3 (

10 3 103 h

10 t

a 60

PI PI PI

2 e 2 2 10 10 D

10 l

l 40 1 1 e 10 1 C 10 10 20

1 2 3 4 1 2 3 4 10 10 10 10 101 102 103 104 10 10 10 10 Unt TNF TNF AnnexinV AnnexinV AnnexinV zVAD Total Cellularity Chemokine Cytokine/Chemokine 12 *** *** 400 7

)

6 6 MIP-2 0 10

300 l 5

MCP-1 RANTES ( m

s /

4 l

KC g ng/ml

200 GMCSF e n 3 8

2 IL-6 l

100 a 1 t o 6

T Unt TNF TNF Unt TNF TNF zVAD zVAD Unt TNF TNF Cytospins zVAD Neutrophils Lymphocytes Monocytes 10 ) 8

6 *** ** *** ** )

)

6 4

*** *** 0

8 (

6 (

(

l 3

6 e

e C 4

C l 2

4 a

o T 2 o 1

2 T

Unt TNF TNF Unt TNF TNF Unt TNF TNF zVAD zVAD zVAD Neutrophils 4 Untreated TNF TNF+zVAD 60 10 104 104 *** ***

)

0 3 3 3 1 45 10 10 ( 1.65 4.74 10 2.11

l l 2 2 e 30 10 10 102

CD11b

CD11b CD11b

a t 101 1 1 o 15 10 10

1 2 3 4 Unt TNF TNF 101 102 103 104 10 10 10 10 101 102 103 104 zVAD Ly6G Ly6G Ly6G SPMs 24 Untreated 4 TNF TNF+zVAD

) 4 4

4 ** * * 10 10 10

( 18 3 3 3

s 10

l 10 10 l 0.53 2.10 0.97

C 12 2 2 2 l 10 10 10

CD11b

CD11b CD11b

o T 6 101 101 101

Unt TNF TNF 1 2 3 4 1 2 3 4 1 2 3 4 zVAD 10 10 10 10 10 10 10 10 10 10 10 10 F4/80 F4/80 F4/80

Inﬁltrate Chemotaxis Lower Chambers

)

d l Lymphocytes Macrophage-like 1.2 o MCP1 f 2.5 *** 300 KC MIP-2 (

150 1.0 x

l d l

n 2.0 *

0.8 I

m / 200 / **

g i g 100

ng/ml n

n 0.6

t 1.5 *** 100 50 0.4 o Neutrophils

0.2 e h 1

Unt IgG Unt Unt IgG IgG IgG Ut SN α KC α KC α KC α KC α MCP-1 α MIP-2 α MCP-1 α MIP-2 α MCP-1 α MIP-2 α MCP-1 α MIP-2 TNF SN

Cell Death and Differentiation Necroptosis suppresses inﬂammation CJ Kearney et al 1325

Cell death assays. Cells were plated at 2 × 105 cells/well in six-well plates and incubated with HRP-conjugated secondary antibody in TBST containing 5% NFDM. treated as indicated 24 h later. Necroptosis was enumerated based on cell After a final 3 × 10 min wash, proteins were detected using West Coast Super Signal morphology (cell swelling, detachment from the plate, membrane rupture) and by (Thermo, Renfrew, UK). flow cytometry-based Annexin V/propidium iodide (PI) staining). A minimum of 300 cells was counted in each treatment. For flow cytometry-based cell death analysis, RNA interference. To ablate mouse RIPK-1, mouse RIPK3 and mouse MLKL cells were harvested by trypsinization and then added to dead cells that expression in L929, MEFs and BMDMs, 5 × 105 cells were transfected with 100 nM were detached from the culture dish. Cells were then incubated with AnnexinV siRNA using nucleofection (Amaxa, Basel, Switzerland) on programme T-020, as (2 μg/ml) and PI (10 μg/ml), followed by aquisition of at least 2500 cells. Necroptotic per the manufacturer’s instructions. Cells were then seeded at 1 × 105 in six-well cell death was confirmed by AnnexinV/PI double positivity, in the absence of plates and incubated with siRNA for 48 h followed by the indicated treatment. siRNA cells with annexinV single positivity. Each assay was repeated a minimum sequences were as follows: mouse RIPK1: sense: 5′-CCACUAGUCUGACUGAUGA of three times. -3′, mouse RIPK3#1: sense: 5′-CCCGACGAUGUCUUCUGUCAA-3′, mouse RIPK3#2: sense: 5′-AAGAUUAACCAUAGCCUUCACCUCCCA-3′, mouse MLKL#1: Measurement of cytokines and chemokines. Cells were plated at sense: 5′-GAGAUCCAGUUCAACGAUA-3′, mouse MLKL#2: sense: 5′-GGAAUAC 2×105 cells/well in six-well plates and treated as indicated 24 h later in 2 ml of CGUUUCAGAUGU-3′. fresh media. Cytokines and chemokines were measured from cell culture supernatants using specific paired antibody ELISA kits obtained from R&D Real-time PCR. HeLa cells were seeded at 5 × 105 in 6-cm dishes. Systems (Abingdon, UK) (human IL-8, human sICAM-1, human RANTES, human The following day, cells were treated as described, and then total RNA was IL-6, human CXCL1, mouse KC, mouse MIP-2, mouse IL1β, mouse RANTES) isolated using an ‘RNeasy’ kit (Qiagen). Normalized RNA (1 μg) was then reverse and eBiosciences (Wycombe, UK) (mouse MCP-1, mouse TNF, mouse IL-6, transcribed using an ‘omniscript’ RT kit (Qiagen). Resulting cDNA was again mouse GMCSF). Generally, antibodies were coated on ELISA plates overnight normalized and used to seed real-time PCR reactions on a ROCHE lightcycler and diluted as per the manufacturer’s instructions in PBS. Samples were then using ‘Fast Start’ (Roche). Actin was used as the control for fold change in incubated on the ELISA plate for 2 h, followed by incubation with biotin-labelled expression. Cytokine and chemokine primers for real-time PCR are included in the detection antibody for 2 h. Samples were then incubated with streptavidin-HRP for Supplementary Information. 30 min prior to detection with TMB substrate. The reaction was stopped with 3 N sulphuric acid, and the absorbance was read on an ELISA plate reader (Tecan, Chemotaxis assays. Chemotaxis assays were performed in Neuro Probe Maennedorf, Switzerland). Each assay was repeated a minimum of three times, (Gaithersburg, MD, USA) 10-Well Chemotaxis Chambers as per the manufacturer’s and all cytokine assays were carried out using triplicate samples from each instructions. Briefly, Supernatants (150 μl) were added to the bottom well of a culture. chemotaxis chamber, and then 8-μM nitrocellulose filters were placed on top and secured by application of the top chamber, to which 200 μl of peritoneal exudate Gene expression analysis by microarray. HeLa cells were seeded at cells were added at 106/ml. Total cells entering the bottom chamber were counted 1×106 in 6-cm plates and treated with TNF (10 ng/ml) 24 h later. After 6 h, cells after 6 h. For antibody-mediated depletion of chemokines, supernatants were were harvested using ‘RNA protect’ (Qiagen, Manchester, UK). RNA extraction, incubated with protein A/G agarose coupled to specific antibodies (1–2 μg/ml) for cDNA synthesis and chip hybridization was carried out by IMGM Laboratories 2 h, followed by depletion of the agarose–antibody–chemokine complexes from the (Martinsried, Germany). supernatants by centrifugation.

Western immunoblotting. To analyse proteins, sodium dodecyl sulphate Animals. Balb/C mice were purchased from Harlan (Blackthorn, UK). Animal polyacrylamide gel electrophoresis was utilized. Protein samples were initially experiments were in accordance with the regulations of the Trinity College Dublin prepared in sample buffer (2% SDS, 50 mM Tris-HCl, pH 6.8, 10% glycerol, 2.5% ethics committee and the Irish Department of Health. β-mercaptoethanol) and then boiled for 7 min to denature proteins. Samples were then loaded into 8–12% polyacrylamide gels and electrophoresed at 75 V. Resolved Statistical analysis. Statistical significance was assessed by unpaired, proteins were then transferred on to nitrocellulose membranes at 40 mA overnight. one-tailed Student’s t-test, in which equal variance was assumed. Error bars Membranes were blocked for 1 h (5% NFDM, 0.05% NaN3 in Tris-buffered saline, represent the mean ± S.E.M. *Po0.1; **Po0.05; ***Po0.01 by Student’s t-test. Tween-20 (TBST)), then incubated with primary antibody and typically diluted at For cytokine/chemokine measurments, triplicate sample denominations were 1 : 1000 for 2 h. Membranes were washed for 3 × 10 min with TBST and then assayed.

Figure 6 Necroptosis attenuates the inflammatory properties of TNF-stimulated cells in vivo.(a) L929 cells at a density of 2 × 106/ml in RPMI supplemented with 1% FCS were pretreated in the presence or absence of zVAD (50 μM), followed by addition of TNF (10 ng/ml). After 24 h, cell death was scored by PI staining, and cytokine/chemokine concentrations in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Supernatants were centrifugated to remove cells or debris. (b) Female Balb/c mice (four per group) were injected intraperitoneally with 300 μl of the indicated L929 cell supernatants from panel (a). After 24 h, mice were killed, and the peritoneal cavity was washed with 4 ml PBS. Total cellularity was scored by microscopy. (c) Cytospins were made from peritoneal-derived cells, stained with hematoxylin/eosin and used to score the percentage of immune cell types by morphology. Population numbers were then generated from total cellularity. (d) Peritoneal infiltrates from panel (b) were immunostained and quantified by flow cytometry as indicated. (e) Supernatants from panel (a) were immunodepleted of the indicated chemokines, and the depletion efficiency was analysed by ELISA. (f) Depleted supernatants from panel (e) were used in a chemotaxis assay using peritoneal infiltrates from mice that received supernatants from TNF-treated L929 cells. Cells from each individual mouse were used as experimental replicates (n = 4). Cytospins were made from cells that successfully migrated, stained with H&E and viewed by phase-contrast microscopy. Error bars represent the mean ± S.E.M. *Po0.1; **Po0.05; ***Po0.01 by Student’s t-test

Figure 7 Necroptosis attenuates LPS-induced inflammation in vivo.(a) Female balb/c mice (4 per group) were injected (intraperitoneally, 200 μl) with PBS or zVAD (250 μg per mouse). After 1 h, mice were injected again with either PBS or LPS (10 μg per mouse). After a further 24 h, mice were killed, and the peritoneal cavity was washed with 4 ml PBS. Total cellularity was scored by microscopy. (b) Spleens were removed, and splenocytes were collected through a strainer. Erythrocytes were removed using RBC lysis buffer and then total cellularity was analysed by microscopy. (c) Peritoneal cell death was analysed by Aqua positivity. (d) Splenocytes were immunostained and analysed by flow cytometry. Population numbers were then generated by multiplying cell percentages by total splenic cellularity counts. Neutrophils (CD11b+, Gr1hi, F4/80med, SiglecF-), eosinophils (SiglecF+), B-cells (CD19+), T-cells(CD3+). (e) Peritoneal-derived cells from control mice were seeded at 1 × 106/ml in 24-well plates. Cells were pretreated with zVAD (20 μM) or not, followed by addition of LPS to 10, 5 and 1 μg/ml. After 48 h, cell death was analysed by forward scatter/PI staining, and cytokine/chemokine concentrations in the culture supernatants were determined by ELISA. Error bars represent the mean ± S.E.M. *Po0.1; **Po0.05; ***Po0.01 by Student’s t-test

Cell Death and Differentiation Necroptosis suppresses inﬂammation CJ Kearney et al 1326

Total PEC Cellularity Total Spleen Cellularity Peritoneal Cell Death 15 80 * *** ** 20

72 ** )

)

1 64

( ( 15 10 (

l l 56

C 48 e

l 10

40 l o 5

T 32 C 5 24 0 0

PBS LPS LPS LPS zVAD PBS zVAD PBS zVAD

LPS+zVAD LPS+zVAD LPS+zVAD Spleen Neutrophils F4/80+ Eosinophils B-Cells T-Cells 3.0 16 5 ** ** 7 **

6 *** ** *

7 6

** * 6 4

0 0

2.5 1

1 3.0 4

1 1 1 12

5 s

l l

l 2.0

l l

e 3

e 4

e e

2.0 c

c c

1.5 8 l

l l 3 l

a 2

t t

1.0 o

o o 2 o

t0 1.0

t0 t t 4 1 0.5 1

LPS PBS LPS PBS LPS PBS LPS PBS LPS PBS zVAD zVAD zVAD zVAD zVAD

LPS+zVAD LPS+zVAD LPS+zVAD LPS+zVAD LPS+zVAD

Peritoneal cells ExVivo Untreated LPS LPS+zVAD zVAD 4 104 104 10 104 36 45 43 53 3 103 103 10 103

2 2 2 2

PI PI 10 10 10 PI 10

1 101 101 10 101 43 36 37 28 200 400 600 800 200 400 600 800 200 400 600 800 200 400 600 800 FSC FSC FSC FSC Peritoneal cells ExVivo 0.6 Necrosis TNF MCP-1 60 2.0 0.5

% 50

(

1.5 l 0.4 40 l

m /

a /

t g

g 0.3

30 1.0 n n

I 0.2

Pe 20 0.5 10 0.1

zVAD - + - + - + - + zVAD - + - + - + - + zVAD - + - + - + - + LPS (μg/ml) LPS (μg/ml) LPS (μg/ml) 50 KC 30 MIP-2 1.4 RANTES

40 25 1.2

l l

m 0.8

/ 30 20

n g

15 g 0.6 n 20 n 10 0.4 10 5 0.2

zVAD - + -+ -+ -+ zVAD - + - + -+ -+ zVAD - + - + - + -+ LPS (μg/ml) LPS (μg/ml) LPS (μg/ml) Figure 7 For caption see previous page

Cell Death and Differentiation Necroptosis suppresses inﬂammation CJ Kearney et al 1327

Conflict of Interest 19. Janes KA, Gaudet S, Albeck JG, Nielsen UB, Lauffenburger DA, Sorger PK. The response The authors declare no conflict of interest. of human epithelial cells to TNF involves an inducible autocrine cascade. Cell 2006; 124: 1225–1239. 20. Burgess AW, Metcalf D. The nature and action of granulocyte-macrophage colony stimulating factors. Blood 1980; 56: 947–958. Acknowledgements. The Martin’s laboratory is supported by SRC (07/SRC/ ’ 21. Brennan FM, McInnes IB. Evidence that cytokines play a role in rheumatoid arthritis. B1144) and PI (08/IN.1/B2031) grants from Science Foundation Ireland. The Lavelle s J Clin Invest 2008; 118: 3537–3545. laboratory is supported by SRC (07/SRC/B1144) and PI grants from Science 22. Kaczmarek A, Vandenabeele P, Krysko DV. Necroptosis: the release of damage-associated Foundation Ireland. SJM is a Science Foundation Ireland Principal Investigator. molecular patterns and its physiological relevance. Immunity 2013; 38:209–223. 23. Zhang Q, Raoof M, Chen Y, Sumi Y, Sursal T, Junger W et al. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 2010; 464: 104–107. 1. Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science 1998; 281: 24. Chen GY, Nuñez G. Sterile inflammation: sensing and reacting to damage. Nat Rev Immunol 2010; 10: 826–837. 1305–1308. 25. Matzinger P. Tolerance, danger, and the extended family. Annu Rev Immunol 1994; 12: 2. Wang L, Du F, Wang X. TNF-alpha induces two distinct caspase-8 activation pathways. 991–1045. Cell 2008; 133: 693–703. 26. Kono H, Rock KL. How dying cells alert the immune system to danger. Nat Rev Immunol 3. Lin Y, Choksi S, Shen HM, Yang QF, Hur GM, Kim YS et al. Tumor necrosis factor-induced 2008; 8: 279–289. nonapoptotic cell death requires receptor-interacting protein-mediated cellular reactive 27. Duprez L, Takahashi N, Van Hauwermeiren F, Vandendriessche B, Goossens V, oxygen species accumulation. J Biol Chem 2004; 279: 10822–10828. Vanden Berghe T et al. RIP kinase-dependent necrosis drives lethal systemic inflammatory 4. Mocarski ES, Upton JW, Kaiser WJ. Viral infection and the evolution of caspase 8-regulated response syndrome. Immunity 2011; 35: 908–918. apoptotic and necrotic death pathways. Nat Rev Immunol 2012; 12:79–88. 28. Vanlangenakker N, Vanden Berghe T, Vandenabeele P. Many stimuli pull the necrotic trigger, 5. He S, Wang L, Miao L, Wang T, Du F, Zhao L et al. Receptor interacting protein kinase-3 an overview. Cell Death Differ 2012; 19:75–86. determines cellular necrotic response to TNF-α. Cell 2009; 137: 1100–1111. 29. Moriwaki K, Ka-Ming Chan F. RIP3: a molecular switch for necrosis and inflammation. Genes 6. Oberst A, Dillon CP, Weinlich R, McCormick LL, Fitzgerald P, Pop C et al. Catalytic activity of the Dev 2013; 27: 1640–1649. – caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis. Nature 2011; 471:363 367. 30. Vercammen D, Beyaert R, Denecker G, Goossens V, Van Loo G, Declercq W et al. Inhibition 7. Dillon CP, Oberst A, Weinlich R, Janke LJ, Kang TB, Ben-Moshe T et al. Survival function of caspases increases the sensitivity of L929 cells to necrosis mediated by tumor – of the FADD-Caspase-8-cFLIP(L) complex. Cell Rep 2012; 1: 401 407. necrosis factor. J Exp Med 1998; 9: 1477–1485. 8. Kaiser WJ, Upton JW, Long AB, Livingston-Rosanoff D, Daley-Bauer LP, Hakem R et al. RIP3 31. Sun X, Lee J, Navas T, Baldwin DT, Stewart TA, Dixit VM. RIP3, a novel apoptosis- – mediates the embryonic lethality of caspase-8-deficient mice. Nature 2011; 471:368 372. inducing kinase. J Biol Chem 1999; 274: 16871–16875. 9. Cho YS, Challa S, Moquin D, Genga R, Ray TD, Guildford M et al. Phosphorylation-driven 32. He S, Liang Y, Shao F, Wang X. Toll-like receptors activate programmed necrosis in assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced macrophages through a receptor-interacting kinase-3-mediated pathway. PNAS 2011; 108: – inflammation. Cell 2009; 137: 1112 1123. 20054–20059. 10. Upton JW, Kaiser WJ, Mocarski ES. Virus inhibition of RIP3-dependent necrosis. Cell Host 33. Yang Y, Tung JW, Ghosn EEB, Herzenberg LA, Herzenberg LA. Division and differentiation – Microbe 2010; 7: 302 313. of natural antibody-producing cells in mouse spleen. PNAS 2007; 104: 4542–4546. 11. Di Paolo NC, Doronin K, Baldwin LK, Papayannopoulou T, Shayakhmetov DM. The 34. Cao C, Lawrence DA, Strickland DK, Zhang L. A specific role of integrin Mac-1 in accelerated transcription factor IRF3 triggers ‘defensive suicide’ necrosis in response to viral and macrophage efflux to the lymphatics. Blood 2005; 106: 3234–3241. bacterial pathogens. Cell Rep 2013; 3: 1480–1486. 35. Linkermann A, Bräsen JH, De Zen F, Weinlich R, Schwendener RA, Green DR et al. 12. Micheau O, Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential Dichotomy between RIP1- and RIP3-mediated necroptosis in tumor necrosis factor- signaling complexes. Cell 2003; 114: 181–190. α–induced shock. Mol Med 2012; 18: 577–586. 13. Van Antwerp DJ, Martin SJ, Kafri T, Green DR, Verma IM. Suppression of TNF-alpha- 36. MacNeill AL, Moldawer LL, Moyer RW. The role of the cowpox virus crmA gene during induced apoptosis by NF-kappaB. Science 1996; 274: 787–789. intratracheal and intradermal infection of C57BL/6 mice. Virology 2009; 384: 151–160. 14. Beg AA, Baltimore D. An essential role for NF-kappaB in preventing TNF-alpha-induced 37. Messud-Petit F, Gelfi J, Delverdier M, Amardeilh MF, Py R, Sutter G et al. Serp2, an inhibitor cell death. Science 1996; 274: 782–784. of the interleukin-1b-converting enzyme, is critical in the pathobiology of myxoma virus. 15. Vince JE, Wong WW, Khan N, Feltham R, Chau D, Ahmed AU et al. IAP antagonists target J Virol 1998; 72: 7830–7839. cIAP1 to induce TNFalpha-dependent apoptosis. Cell 2007; 131: 682–693. 38. Robinson N, McComb S, Mulligan R, Dudani R, Krishnan L, Sad S. Type I interferon induces 16. Esche C, Stellato C, Beck LA. Chemokines: key players in innate and adaptive immunity. necroptosis in macrophages during infection with Salmonella enterica serovar Typhimurium. J Invest Dermatol 2005; 125: 615–628. Nat Immunol 2012; 13: 954–962. 17. Rot A, Von Andrian UH. Chemokines in innate and adaptive host defense: basic 39. Abtin A, Jain R, Mitchell AJ, Roediger B, Brzoska AJ, Tikoo S et al. Perivascular chemokinese grammar for immune cells. Ann Rev Immunol 2004; 22: 891–928. macrophages mediate neutrophil recruitment during bacterial skin infection. Nat Immunol 18. Rollins BJ. Chemokines. Blood 1997; 90: 909–928. 2014; 15:45–53.

Supplementary Information accompanies this paper on Cell Death and Differentiation website (http://www.nature.com/cdd)

Cell Death and Differentiation