Caspase-8 mediates caspase-1 processing and innate immune defense in response to bacterial blockade of NF- B and MAPK signaling

Abstract

Significance
Pathogenic organisms express virulence factors that can inhibit immune signaling pathways. Thus, the immune system is faced with the challenge of eliciting an effective inflammatory response to pathogens that actively suppress inflammation. The mechanisms that regulate this response are largely undefined. The Yersinia virulence factor YopJ blocks NF-κB and MAPK signaling, resulting in reduced cytokine production and target cell death. Here, we find that caspase-8, RIPK1, and FADD are required for YopJ-induced cell death and show that mice lacking caspase-8 are severely susceptible to Yersinia infection and have defective proinflammatory cytokine production. These findings highlight a possible mechanism of immune defense that can overcome pathogen inhibition of cell-intrinsic proinflammatory immune responses.

Full text

Caspase-8 and RIP kinases regulate bacteria-induced
innate immune responses and cell death
Dan Weng
a
, Robyn Marty-Roix
a
, Sandhya Ganesan
a
, Megan K. Proulx
b
, Gregory I. Vladimer
a
, William J. Kaiser
c
,
Edward S. Mocarski
c
, Kimberly Pouliot
a
, Francis Ka-Ming Chan
d
, Michelle A. Kelliher
e
, Phillip A. Harris
f
, John Bertin
f
,
Peter J. Gough
f
, Dmitry M. Shayakhmetov
g
, Jon D. Goguen
b
, Katherine A. Fitzgerald
a,h
, Neal Silverman
a
,
and Egil Lien
a,h,1
a
Program in Innate Immunity, Division of Infectious Diseases and Immunology, Department of Medicine,
b
Department of Microbiology and Physiological
Systems,
d
Department of Cancer Biology, and
e
Department of Pathology, University of Massachusetts Medical School, Worcester, MA 01605;
c
Department of
Microbiology and Immunology, Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322;
f
Pattern Recognition Receptor Discovery
Performance Unit, Immuno-inflammation Therapeutic Area, GlaxoSmithKline, Collegeville, PA 19426;
g
Lowance Center for Human Immunology, Departments
of Pediatrics and Medicine, Emory University, Atlanta, GA 30322; and
h
Centre of Molecular Inflammation Research, Department of Cancer Research and
Molecular Medicine, Norwegian University of Science and Technology, 7491 Trondheim, Norway
Edited by Ruslan Medzhitov, Yale University School of Medicine, New Haven, CT, and approved April 1, 2014 (received for review February 25, 2014)
A number of pathogens cause host cell death upon infection, and
Yersinia pestis, infamous for its role in large pandemics such as the
“Black Death”in medieval Europe, induces considerable cytotoxic-
ity. The rapid killing of macrophages induced by Y. pestis, depen-
dent upon type III secretion system effector Yersinia outer protein
J (YopJ), is minimally affected by the absence of caspase-1, cas-
pase-11, Fas ligand, and TNF. Caspase-8 is known to mediate apo-
ptotic death in response to infection with several viruses and to
regulate programmed necrosis (necroptosis), but its role in bacte-
rially induced cell death is poorly understood. Here we provide
genetic evidence for a receptor-interacting protein (RIP) kinase–
caspase-8-dependent macrophage apoptotic death pathway after
infection with Y. pestis, influenced by Toll-like receptor 4-TIR-do-
main-containing adapter-inducing interferon-β(TLR4-TRIF). Inter-
estingly, macrophages lacking either RIP1, or caspase-8 and RIP3,
also had reduced infection-induced production of IL-1β, IL-18, TNF,
and IL-6; impaired activation of the transcription factor NF-κB; and
greatly compromised caspase-1 processing. Cleavage of the proform
of caspase-1 is associated with triggering inflammasome activity,
which leads to the maturation of IL-1βand IL-18, cytokines impor-
tant to host responses against Y. pestis and many other infectious
agents. Our results identify a RIP1–caspase-8/RIP3-dependent cas-
pase-1 activation pathway after Y. pestis challenge. Mice defective
in caspase-8 and RIP3 were also highly susceptible to infection and
displayed reduced proinflammatory cytokines and myeloid cell death.
We propose that caspase-8 and the RIP kinases are key regulators of
macrophage cell death, NF-κB and inflammasome activation, and host
resistance after Y. pestis infection.
The causative agent of plague, Yersinia pestis is well known to
cause significant cell death upon infection (1–3). Like the
activation of inflammatory pathways to produce cytokines, trig-
gering cell death pathways is a common response of the mam-
malian immune system to infection. Death of immune cells can
eliminate the replication niche of pathogens found within those
cells, thus inhibiting the proliferation of the pathogens and ex-
posing them to bactericidal mechanisms (4). Conversely, elimi-
nation of key immune cells can diminish the ability of those cells
to respond to infection. Multiple host and microbial factors control
cell death pathways (5). Caspase-8–dependent apoptosis, receptor
interacting protein-1 (RIP1)- and RIP3-dependent necroptosis, and
caspase-1/caspase-11–dependent pyroptosis constitute major modes
of regulated cell death during infection (5, 6). Several viruses seem to
induce caspase-8–dependent apoptosis (7). Caspase-8 has also been
suggested to have additional functions, such as inhibiting necroptosis
(7–9) and modulation of NF-κBactivationinTandBcells(10).
Signaling to the transcription factor NF-κB controls the transcription
of cytokines such as IL-6, TNF, pro-IL-1β, and pro-IL-18, and
stimulates cell survival. Y. pestis can induce cell death in macrophages
and dendritic cells via the type III secretion system (T3SS) effector
Yersinia outer protein J (YopJ; YopP in Yersinia enterocolitica), al-
though it is unclear whether this is entirely by apoptosis (11, 12). All
human-pathogenic Yersiniae (Y. pestis,Yersinia pseudotuberculosis,
and Y. enterocolitica) harbor cytotoxic properties toward host cells,
and YopJ production is associated with cell death in vivo and in
vitro (13–16). YopJ-mediated inhibition of NF-κB by acetylation of
Inhibitor of κBKinaseβ(IKKβ), MAP kinase kinases, and TAK1
may modulate macrophage death via effects on inflammatory and
prosurvival signals (2, 17–21). Inflammasome activation, culminat-
ing in the activation and processing of caspase-1, leads to the pro-
duction of IL-18 and IL-1β, key inflammatory cytokines and
antibacterial defenses, but can also be associated with caspase-1–
dependent pyroptotic cell death (22). YopJ also participates in
inflammasome activation (16, 23), leading to a host immune re-
sponse. Thus, this single bacterial effector may induce both pro-
tective and harmful effects for the host. In the present study we
investigated the mechanisms for Y. pestis-induced cell death, NF-κB
activation, and triggering of inflammasome activation.
Results and Discussion
Yersinia Induces Cell Death via RIP1, Caspase-8, and RIP3. Viable
Y. pestis KIM5 can induce rapid cell death via YopJ (Fig. S1A).
Rapid death in bone marrow-derived macrophages (BMDMs) is
induced in a YopJ-dependent manner by Y. pestis or Y. pseudo-
tuberculosis temperature-shifted from 26 °C to 37 °C (Fig. S1 A
Significance
Receptor-interacting protein-1 (RIP1) kinase and caspase-8 are
important players in activation of apoptotic pathways. Here
we show that RIP1, caspase-8, and RIP3 contribute to infection-
induced macrophage cell death and also are required for acti-
vation of transcription factor NF-κB and caspase-1 upon in-
fection with the bacterial pathogen Yersinia pestis, the causative
agent of plague. Mice lacking caspase-8 and RIP3 are also very
susceptible to bacterial infection. This suggests that RIP1, cas-
pase-8, and RIP3 are key molecules with multiple roles in innate
immunity during bacterial challenge.
Author contributions: D.W., R.M.-R., and E.L. designed research; D.W., R.M.-R., G.I.V., and
E.L. performed research; S.G., M.K.P., W.J.K., E.S.M., K.P., F.K.-M.C., M.A.K., P.A.H., J.B.,
P.J.G., D.M.S., J.D.G., K.A.F., and N.S.contributed new reagents/analytic tools; D.W., R.M.-R.,
K.A.F., N.S., and E.L. analyzed data; and D.W., R.M.-R., J.D.G., K.A.F., N.S., and E.L. wrote
the paper.
Conflict of interest statement: E.L. and J.D.G. have a patent application on the use of
modified bacteria as used in vaccines. P.A.H., J.B., and P.J.G. are employees and share-
holders of GlaxoSmithKline.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: Egil.Lien@umassmed.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1403477111/-/DCSupplemental.
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IMMUNOLOGY

and B), a condition that mimics the temperature change associated
with infection via a fleabite. In addition to arming the T3SS, the
temperature shift ensures the initial presence of some TLR4-
stimulatory LPS (Fig. S1C) (24). Although caspase-1 is activated
by Y. pestis (1, 25), macrophage death was independent of cas-
pase-1/caspase-11, suggesting nonpyroptotic cytotoxicity (Fig. S2
Aand B). Death was unaffected by Fas ligand (FasL) or TNF,
indicating that those death receptor-mediated mechanisms are
not involved; and independent of inflammasome-related NOD-
like receptors (NLRs), the RNA-dependent protein kinase
(PKR), the inflammasome adaptor apoptosis-associated speck-
like protein containing a CARD (ASC), IL-1β, and IL-18 (Fig.
S2 C–G) (1). Caspase-8 is a key enzyme in cell death induced by
some viruses (26). Caspase-8 deficiency results in embryonic le-
thality, but mice deficient in both caspase-8 and RIP3 [RIP3
−/−
caspase-8
−/−
mice, double knockout (dKO)] are rescued. These
data indicate a vital role for caspase-8 in suppressing necroptosis
by targeting a component of the RIP3 pathway (8, 9). Macro-
phages from RIP3
−/−
caspase-8
−/−
mice were remarkably resistant
to cell death induced by Y. pestis and Y. pseudotuberculosis, but not
by Salmonella, which induces pyroptotic death (4), or with the
NLRP3 inflammasome-specific trigger nigericin (Fig. 1 Aand B).
YopJ-induced death is likely not necroptosis because RIP3-
deficient cells are not protected (Fig. 1 Aand B). Electron
microscopy revealed that macrophages infected with Y. pestis
displayed features consistent with apoptotic death, such as
membrane blebbing and nuclear condensation and fragmen-
tation. These effects were absent in visibly infected dKO cells (Fig.
1C). Moreover, infection of macrophages with Y. pestis led to
DNA fragmentation patterns typically associated with apopto-
sis, and this was blocked by zVAD pan-caspase inhibition (Fig. S3A).
Taken together, our data strongly suggest that Yersinia induces
rapid macrophage death by apoptosis via caspase-8.
RIP3-mediated necroptotic death requires RIP1 (27–29), a
serine/threonine kinase that canalso contribute to NF-κB signaling
(30) and apoptosis. RIP1
−/−
mice die shortly after birth (31), but
fetal liver macrophages from RIP1
−/−
mice, in contrast to RIP3
−/−
macrophages, displayed a rescue from death induced by Y. pestis
(Fig. 2A) and DNA laddering (Fig. S3C), suggesting that RIP1
activity contributes to apoptotic cell death upon infection, likely
mediated by the induction of caspase-8 enzymatic activity and
cleavage of procaspase-8 that precedes cell death (Figs. 1D,and2A,
B,E,andFand Fig. S3 Dand E). RIP1
−/−
macrophages were also
protected from necroptotic cell death induced by heat-killed
KIM5 plus zVAD or LPS plus zVAD (Fig. S3F). Potent and speci-
fic inhibitors of RIP3 (32) or RIP1 [GlaxoSmithKline (GSK): P.A.H.,
J.B., P.J.G.] kinase activity have recently been identified. The
RIP1 inhibitor GSK’963, but not inactive enantiomer GSK’962,
blocks Y. pestis-induced cell death (Fig. 2Cand Fig. S3B) and
caspase-8 activity (Fig. 2D). In addition to the genetic and phar-
macological interactions between RIP1 and caspase-8, we found
that RIP1 biochemically interacted with caspase-8 after Y. pestis
challenge (Fig. 2E). Cell death, cleavage of procaspase-8, and
enzymatic activity were partially reduced in the absence of TLR4
and TRIF, but not MyD88 (Fig. S4). Reduced death was also seen
for bacteria grown at 37 °C and Y. pestis-EcLpxL, which consti-
tutively generates a hexa-acylated LPS (Fig. S5 Aand B). TLR4
signaling seems to enhance early caspase-8–mediated effects by Y.
pestis YopJ, similar to those proposed for Y. enterocolitica YopP
(33–35). Cell death induced by Y. pestis grown at 37 °C was
inhibited by the presence of CaF1 capsule protein (Fig. S5 Cand
D), suggesting that the capsule prevented close contact between
bacteria and host cells needed for T3SS effects.
The targeted deletion of caspase-8 in myeloid cells [condi-
tional KO (cKO) caspase-8
fl/fl
LysM cre
+/+
generated by D.M.S.;
Fig. S6 Aand B] had little effect on Y. pestis-induced macrophage
death (Fig. 2C). Although the generation of other mice with
defects in caspase-8 in macrophages has been reported (36), our
caspase-8 cKO BMDM appeared healthy in culture and did not
display increased cell death in the presence or absence of infection
(Fig. 2C). Blockade of RIP3 kinase activity with GSK’872 strongly
reduced macrophage death in the absence of caspase-8, suggesting
that deletion of caspase-8, or caspase inhibition by zVAD (Fig. 2C
and Fig. S6C), may promote necroptosis by RIP3, presumably
influenced by reduced Y. pestis-induced cleavage of RIP1 in the
absence of caspase-8 (Fig. 2F).
Cleavage and activation of the downstream apoptotic execu-
tioner caspase-3 was also dependent upon YopJ and caspase-8–
RIP3 (Fig. S6D). The caspase-8–RIP3 pathway also influenced
death induced by Y. enterocolitica but not by Salmonella or
Pseudomonas, which also harbor a T3SS (Fig. S6E). Thus, all
human-pathogenic Yersiniae, but not all bacteria containing a
T3SS, trigger cell death via the same pathway. Our results pro-
vide an explanation for how Yersinia induces macrophage cell
death via caspase-8 and RIP kinases. In this model, caspase-8–
dependent apoptosis represents the default, whereas caspase-8
absence may lead to RIP3-dependent necroptosis. RIP1 has a key
upstream role for both modes of death, perhaps influenced by its
ability to direct apoptosis under conditions of cIAP1 depletion
(37) as seen with Y. pestis (Fig. S6F).
Effects on NF-κB Activity. Caspase-8 has also been suggested to
regulate NF-κB activity (10, 38, 39). We found a reduction in TNF
and IL-6 release, and pro-IL-1βexpression, all controlled by NF-
κB, in RIP1 KO, caspase-8 cKO, and RIP3
−/−
caspase-8
−/−
, but not
in RIP3
−/−
macrophages upon infection or LPS treatment (Fig. 3
A–F). However, cytokine production by the TLR2 ligand Pam3Cys
(Fig. 3 Aand B) or Sendai virus (Fig. S7A) was largely preserved.
The defect in cytokine release could be explained by a decreased
NF-κB activation, as suggested by reduced IκBαdegradation,
IκBαphosphorylation, IKKα/βphosphorylation, and p65 nuclear
translocation, particularly at later time points during Y. pestis
or LPS challenge (Fig. 3 G–Jand Fig. S7 Band D). Reduced
signaling could also be observed in RIP1
−/−
macrophages (Fig. 3E
WT, uninfected WT, Y. pess RIP3- /-Casp8 -/-,Y.pess
C57Bl/6
RIP3-/-Casp8-/-
RIP3-/-Casp8+/-
RIP3-/-
0
20000
40000
60000
80000
100000 Medium
Y. pestis
BA
C
D
Salmonella
LPS+Nigericin
0
20
40
60
80
100
**
0
20
40
60
80 WT
RIP3-/-
RIP3-/-Casp8-/-
**
**
Y.pestis
Y.pseudo.
Caspase 8 activity (RLU)
*
*
Y.pestis
% cell death
Fig. 1. Caspase-8–RIP3-deficient macrophages are protected against Y. pestis
induced cytotoxicity. (Aand B) Caspase-8
−/−
RIP3
−/−
(dKO), but not RIP3 KO
BMDM, are protected from Yersinia-induced cytotoxicity measured by LDH
release assay or (C) electron microscopy. (Scale bars, 2 μm.) Asterisks in Cin-
dicate bacteria. (D) Caspase-8 activity induced by Y. pestis infection (MOI 40,
2 h) in WT, RIP3
−/−
and dKO BMDMs. BMDM were infected with 10–40 MOI of
Yersiniae or 1.5 MOI of Salmonella typhimurium for 4 h (Aand B)or2h(C
and D), and gentamycin was added after 1 h. Figures are representative for
three to eight experiments performed. Bars indicate mean plus SD. **P<0.01
(two-tailed ttest).
7392
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www.pnas.org/cgi/doi/10.1073/pnas.1403477111 Weng et al.

and Fig. S7E). How caspase-8 controls NF-κB activation is unclear
and may not involve the enzymatic activity of caspase-8 (39) (Fig.
S7C); However, TRIF-mediated pathways may be targeted be-
cause MyD88-dependent TLR2 signaling is not affected.
Subsequent experiments indicated that YopJ-dependent
Y. pestis-induced IL-1βor IL-18 release was reduced in the ab-
sence of caspase-8 and showed further reduction by the absence
or blockade of RIP3 or RIP1 kinase activity (Fig. 4 A–E).
B
A
Y.pestis
Salmonella
0
50
100
150
% c ell d eath
RIP1+/+
RI P1-/ -
**
**
RIP1+/+
RIP1-/-
0
20000
40000
60000
80000 Medium
Y.pestis
Caspase 8 activity (R LU)
No Ab
Medium
Y.pestis
∆YopJ
IP, RIP1 Ab
Caspase-8
RIP1
*
D
C
RIP1 INH GSK’963
CTR GSK’962
RIP3 INH GSK’872
-
-
-
+
-
-
-
+
-
-
-
+
Y.pestis
0
20
40
60
80 WT
% c el l deat h
Caspase 8 CKO
**
**
0
20000
40000
60000
80000
Caspase 8activity (RLU)
RIP1 INH GSK’963 --
+
Y.pestis
**
EF
Medium
Y.pestis
Medium
Y
.pestis
Medium
Y.pestis
Medium
Y.pestis
WT DKO RIP3-/- Casp8 CKO
RIP1
RIP3
β-actin
-70KD
-35KD
Fig. 2. RIP1 inhibition or deficiency protect macrophages from Y. pestis-induced cell death. (A) RIP1-deficient fetal liver macrophages are resistant to Y. pestis-
induced killing (MOI 40, 4 h), detected by LDH release. (Band D) RIP1, but not RIP3, mediates caspase-8 enzymatic activity after infection of BMDM (D) or fetal
liver macrophages (B) with Y. pestis for 2 h. (C) Caspase-8 conditional KO macrophages are protected from Y. pestis-induced death in the presence of RIP1
(GSK’963) or RIP3 (GSK’872) kinase inhibitors, but not by inactive compound GSK’962. (D) RIP1 kinase inhibitor GSK’963 inhibits caspase-8 enzyme activity after
infection. (E) RIP1 forms a complex with caspase-8 upon infection (1 h), measured by co-IP. (F) RIP1 is cleaved after Y. pestis infection in a caspase-8 dependent
fashion. Figures are representative for three to eight experiments performed. Bars indicate mean plus SD. **P<0.01 (two-tailed ttest).
ns
***
*
Medium
0
2
4
6
8
10
WT
Caspase 8 CKO
*
**
**
**
**
ΔΔ Δ
WT RIP3-/-Casp8-/- RIP3-/-
pro IL-1β-
β-actin-
Un Y.pe s t i s YopJ Un Y.p e s ti s YopJ Un Y. p e s ti s YopJ
0 15 30 45 0 15 30 45
(min)
WT RIP3-/-Casp8-/-
p-IĸBα
IĸBα
β-actin
Y.pestis ΔYopJ
0 15 30 45 0 15 30 45
WT RIP3-/-Casp8-/-
LPS (min)
pIKKα/β
IKKα
β-actin
B
EF
G
H
I
J
AC
D
IκBα
β-actin
WT RIP3-/-Casp8-/- RIP3-/-
0 15 30 45 60 80 0 15 30 45 60 80
WT RIP3-/-Casp8-/-
p-IκBα
IκBα
β-actin
LPS (min)
**
*
Y. pestis
Medium
Pam3Cys
LPS
Y.pestis
Salmonella
0
5
10
15
20 RIP1+/+
RIP1-/-
IL-6 (ng/mL)
Medium
Y.pestis
LPS
0
5
10
15
WT
RIP3-/-Casp8-/-
IL-1βmRNA
(Arbitrary units)
ns
**
Medium
0
1
2
3
4WT
RIP3-/-
RIP3-/- Casp8-/-
Y. pestis
Pam3Cys
0
2
4
6
8WT
RIP3-/-
RIP3-/- Casp8-/-
Y. pestis
Pam3Cys
Medium
LPS
0
5
10
15
IL-6 (ng/mL)
Medium
0
1
2
3
4
TNFα(ng/mL)
IL-6 (ng/mL)
Medium
Y. pestis
Y. pseudo.
LPS
Fig. 3. Caspase-8 and RIP1 contribute to cytokine
release and NF-κB activation. (A–C) WT or mutant
BMDM were infected with Y. pestis,Y. pseudotu-
berculosis (MOI 10), or Salmonella (Sal, MOI 1.5) or
treated with LPS (100 ng/mL) or Pam3Cys (500 ng/
mL) for 6 h, and cytokine release was measured by
ELISA. (D) BMDMs were infected with Y. pestis for
4 h, mRNA was isolated, and quantitative PCR for
pro-IL-1βwas performed. (E) WT or RIP1
−/−
fetal
liver macrophages were stimulated with LPS (50
ng/mL), Pam3Cys (500 ng/mL), Y. pestis (MOI 10),
or Salmonella (MOI 1.5) for 6 h. IL-6 release was
measured by ELISA. (F) BMDMs were infected for
6 h and cell lysates probed for pro-IL-1β.(G–J)
BMDMs were infected or treated with LPS, mouse
TNF-α(10 min), and cell lysates were probed by
immunoblot for the indicated proteins (IκBα,
phospho-IκBα, phospho-IKKα/β,orβ-actin). Figures
are representative of two to five experiments
performed. Bars indicate means plus SD. **P<
0.01, *P<0.05 (two-tailed ttest).
Weng et al. PNAS
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IMMUNOLOGY

Cytokine release after stimulation with Pam3Cys and nigericin
was unaffected (Fig. 4 Aand B), implying that NLRP3 activation
was not decreased. Although the absence of caspase-8 alone in
macrophages decreased IL-1βrelease induced by infection, it
increased IL-1βinduced by LPS alone (Fig. 4E), as suggested for
dendritic cells (40). Thus, more complex stimulations, as ob-
served during infection, yield a different result than a purified
ligand, possibly reflecting combined effects induced by both LPS
and the Yersinia T3SS in the context of live bacteria.
RIP1, Caspase-8, and RIP3 Mediate Inflammasome Activation. Our
previous data (Fig. 3) could partially explain reduced IL-1β
release. However, caspase-8 or RIP1 inhibition minimized
infection-induced caspase-1 cleavage (Fig. S7 Fand G), in-
dicating direct effects on inflammasome action. RIP3 has
been involved in inflammasome activation under certain con-
ditions with cIAP inhibiton (41). Infection-induced caspase-1 pro-
cessing, only partially dependent upon NLRP12 (25), was not
affected in RIP3
−/−
cells but was reduced in TLR4 or TRIF KO
(Fig. S8 Aand B) and caspase-8 cKO cells, and severely reduced in
RIP1
−/−
or RIP3
−/−
caspase-8
−/−
cells after Y. pestis infection (Fig. 5
A,B,andD). IL-1βprocessing was also affected (Fig. 5 Cand D).
Caspase-1 cleavage induced by Salmonella and Pam3Cys plus
nigericin was not affected (Fig. 5 Aand B), indicating that NLRC4-
and NLRP3-mediated caspase-1 cleavage is not inherently reduced
in RIP1
−/−
or dKO cells. Caspase-8 has been proposed to control
IL-1βmaturation and release in response to FasL stimulation or
fungal and bacterial challenge (42, 43), perhaps by directly cleaving
pro-IL-1β(44), and we cannot exclude this possibility. However, we
propose that caspase-8 directs caspase-1 processing and activation,
in a RIP3-enhanced manner, after Y. pestis challenge (Fig. 5 Band
D), but caspase-1 does not control caspase-8 activation (Fig. S8C).
Caspase-8 may be a critical component, but deletion or inhibition of
RIP3 may block an alternative pathway in the absence of caspase-8,
redundancy between caspase-8 and RIP3 may occur, or both mol-
ecules may be needed for stabilization of a signaling complex. The
mechanism we describe seems independent of FasL, TNF, or type
IIFN(Fig. S8 D–F) and may have some common features with
responses induced by ER stress, certain chemotherapeutic drugs, or
Citrobacter (45–47). The effect on caspase-1 cleavage may be me-
diated by the inflammasome adaptor ASC (Fig. 5E), because ASC
can associate with caspase-8 after Francisella or Salmonella in-
fection (38, 48), although the role of ASC may differ depending
upon conditions and source of YopJ (16, 23, 25).
Role of Caspase-8 and RIP3 for in Vivo Resistance to Bacterial
Infection. The in vivo relevance of our findings was emphasized
by the fact that RIP3
−/−
caspase-8
−/−
mice were more susceptible
to s.c. infection with virulent Y. pestis KIM1001 (Fig. 6A). Be-
cause LD
50
is very low for KIM1001 we used the attenuated
strain KIM1001-EcLpxL, which constitutively generates a TLR4-
activating hexa-acylated LPS (24), for survival analysis. dKO, or
lethally irradiated WT mice with bone marrow transplant (BMT)
from dKO, succumbed to s.c. infection with Y. pestis-EcLpxL
(Fig. 6Band Fig. S9A). Resistance to Y. pestis-LpxL is heavily
influenced by IL-18 and IL-1 (25). Moribund mice had large
numbers of bacteria in their spleens compared with WT controls,
suggesting that death occurred from uncontrolled systemic bac-
terial replication (Fig. 6C). This correlated with depressed IL-18,
IL-1β, TNF, and IL-6 cytokine levels and reduced myeloid cell
death (cells positive for live/dead stain and annexin V) in spleens
(Fig. 6 D–Iand Fig. S9 B–G) after i.v. infection. Reduced ability
to suppress bacterial growth was also suggested by the presence
of visible bacteria-containing pockets in inflammatory foci in the
livers (Fig. 6J) of dKO BMT mice upon i.v. infection. Because
irradiated mice that received RIP3
−/−
caspase-8
−/−
BMT behaved
similarly as dKO animals, we propose that protection toward
infection is mediated by cells originating from the bone marrow,
expressing caspase-8 and RIP3. Some questions still remain with
respect to certain details of how caspase-8 and RIP3 are involved
in caspase-1 processing, although it is possible that ASC has
a central role. Our results provide a basis for increased un-
derstanding of how bacterial pathogens, via their T3SS, can in-
teract with several aspects of host innate immunity via RIP
kinases and caspase-8. The data also show how apoptosis, gen-
erally viewed as a “silent”cell death, can be accompanied by
strong inflammatory reactions, via pathways with several com-
mon players. The host may have developed these pathways as an
effective means of alerting cells to the infection. We propose that
caspase-8 and RIP kinases are central regulators of cell death
and innate immune responses to Y. pestis, and we establish a role
for these components in antibacterial innate immune responses.
Therapies that modulate the activity of these pathways may be
useful in the treatment of bacterial infections.
Methods
Mice. RIP3 KO (49) and caspase-8
−/−
RIP3
−/−
(dKO) (9) have been reported.
Caspase-8
fl/fl
LysM cre
+/+
cKO mice were generated by D.M.S. C57BL/6 mice
were bred in house or from Jackson Laboratories. BMT was performed on
lethally (900 rads) irradiated mice. Mice were infected s.c. or i.v. with 500 cfu
of KIM1001-pEcLpxL and monitored for survival. Tissue for analysis was
harvested at 42 h after infection, or at 68 h after s.c. infection with KIM1001
(300 cfu).
Bacterial Strains and Growth Conditions. Y. pestis KIM5 or KIM5ΔYopJ (24)
(25) were grown in tryptose-beef extract broth with 2.5 mM CaCl
2
overnight
with shaking at 26 °C. The next day the bacteria was diluted 1:8 in fresh
media, cultured for 1 h at 26 °C, and shifted to 37 °C for 2 h or grown
continuously at 37 °C when indicated. Y. pseudotuberculosis IP2666,
Y. enterocolitica 8081, and Salmonella enterica serovar Typhimurium strain
SL1344 were as reported (25) and grown at 37 °C. KIM5-EcLpxL and
KIM1001-EcLpxL were as previously published (24).
**
Medium
Y. pestis
Y. pestis∆YopJ
0.0
0.5
1.0
1.5
WT
RIP1 INH GSK’963 ---+
RIP3 INH GSK’872 --+-
Y.pestis
**
**
**
***
LPS
0
200
400
600
800
1000 WT
Caspase 8 CKO
** **
**
B
A
CDE
WT
RIP3-/-
RIP3-/-Casp8-/-
IL-1β (pg/mL)
0.0
0.2
0.4
0.6
0.8
1.0
5
10
15
WT
Caspase 8 CKO
**
Medium
Y. pestis
Pam3+Nigericin
IL-1β (ng/mL)
Caspase 8 CKO
IL-1β (ng/mL)
IL-18 (pg/mL)
Medium
Y. pestis
Y. pestis∆YopJ
IL-1β (pg/mL)
Medium
Y. pestis
LPS+Nec-1
***
ns
Medium
Pam3+Nigericin
IL-1β (ng/mL)
0.0
0.5
1.0
1.5
2.0
2.5
4
6
8
10
Y. pestis
0
100
200
300
400
0
50
100
150
200 WT
RIP3-/-
RIP3-/-Casp8-/-
Fig. 4. Y. pestis-induced release of IL-1βand IL-18 is severely reduced in
caspase-8/RIP3-deficient macrophages. (A–E) BMDMs were infected with
Y. pestis or Y. pestis ΔYopJ for 6 h as indicated in Fig. 1, or stimulated with
nigericin (10 μg/mL) for 1 h after priming with Pam3Cys (4 h, 500 ng/mL).
IL-1βand IL-18 were analyzed by ELISA. (C) Some BMDMs were treated with
RIP1 inhibitor GSK’963 (1 μM) or RIP3 inhibitor GSK’872 (10 μM) for 1 h be-
fore infection. (E) BMDMs were challenged with Y. pestis (MOI 10) for 6 h or
LPS (50 ng/mL) for 10 h with or without Nec-1 pretreatment (20 μM). Figures
are representative of three to five experiments. Bars indicate means plus SD.
**P<0.01, *P<0.05 (two-tailed ttest in A,B, and D, and two-way ANOVA
with Tukey’s posttest in Cand E).
7394
|
www.pnas.org/cgi/doi/10.1073/pnas.1403477111 Weng et al.

Cell Stimulations. BMDMs were prepared by maturing bone marrow cells for6–
7 d in the presence of L929 supernatant containing M-CSF. Some experiments
were performed with BMDM immortalized with J2 retrovirus (42), or J2 im-
mortalized RIP1
+/+
and RIP1
−/−
fetal liver macrophages (31). Cells were plated
overnight and infected with bacteria at multiplicities of infection (MOIs) of 10
or 40, or stimulated with LPS from Y. pestis 26 °C (24) or Escherichia coli,or
Pam3Cys (Invivogen). Gentamycin was added 1–2 h after infection. Cell death
wasestimatedat4hbymeasuringlactate dehydrogenase (LDH) release
(Promega). In some experiments, cells were pretreated with 1 μMGSK’963
or GSK’962, or 3 μMGSK’872 [RIP1 and RIP3 inhibitors (32) and GSK: P.A.H.,
J.B., P.J.G.], 20 μM Nec-1 (Enzo), 20 μM zIETD, zYVAD, or zVAD (Promega)
for 1 h before infection. Cytokines and caspase-1 cleavage were measured
Medium Y. p e s ti s Y.e n t e r c o
.Sal
.
Pam3+Nigericin
Pro Caspase-1
Caspase-1 p20
Lysate
Pro Caspase-1
SN
WT DKO RIP3-/- WT DKO RIP3-/- WT DKO RIP3-/-
Y. p e st i s S a l Pam3+Nigericin
Pro Caspase-1
Caspase-1 p20
SN
Lysate
WT DKO RIP3-/-
Medium
Pro Caspase-1
β-actin
WT DKO RIP3-/-
WT DKO RIP3-/- WT DKO RIP3-/-
Medium Y.pestis Pam3+Nigericin
-IL-1β p17
-pro IL-1β
SN
Lysate
-β-actin
Medium
Y.pestis
Pam3+Y.pestis
Medium
Y
.pestis
Pam3+Y.pestis
WT Caspase 8 CKO
Pro IL-1β
Pro IL-1β
IL-1β p17
Pro Caspase-1
Pro Caspase-1
Caspase-1 p20
β-actin
Medium
Y.pestis
Pam3+Niger.
Medium
Y.pestis
Pam3+Niger.
WT ASC-/-
Pro Caspase-1
Caspase-1 p20
AB
C
D
E
Fig. 5. RIP kinases and caspase-8 control caspase-1 cleavage induced by Y. pestis.(A–E) BMDM (WT, RIP3
−/−
, RIP3
−/−
caspase-8
−/−
dKO, caspase-8 cKO) or fetal
liver macrophages (RIP1
+/+
, RIP1
−/−
) were infected with Y. pestis,Y. enterocolitica,orSalmonella (Sal) for 6 h or primed with Pam3Cys followed by nigericin for
1 h, and supernatants (SN) or lysates were analyzed for caspase-1 or IL-1βprocessing by immunoblots. Figures are representative of three to five experiments.
WT RIP3-/-Casp8-/-
WT
RIP3-/-Casp8-/-
1
2
3
4
5
6
7
8
9
10
**
Detection limit
WT RIP3-/-Casp8-/-
Annexin V
Live/Dead Blue
Y.pestis infected
2.05% 25.4%
9.56%62.9%
11.8%0.656%
7.35%80.2%
WT
RIP3-/-Casp8-/-
0
10
20
30
40
% of CD11b +Cel ls
Annexin V+, Live/Dead Blue+
Uninfected
WT
0
20
40
60
80
IL-1β(ng/g)
Uninfected
WT
RIP3-/-Casp8-/-
0.00
0.75
1.50
40
80
IL-6 (ng/g)
**
AB D
EFG I
H
C
J
WT
2
3
4
5
6
7
8
9
10
RIP3-/-Casp8-/-
CFU/spleen (log)
*
*
***
*
CFU/spleen (log)
RIP3-/-Casp8-/-
0
200
400
600
800
TNFa (pg/g)
Uninfected
WT
RIP3-/-Casp8-/-
0.0
0.5
1.0
25
50
IL-18 ( ng/g)
**
Uninfected WT
Uninfected DKO
WT
RIP3-/-Casp8-/-
p<0.001
Day
0 5 10 15 20
0
20
40
60
80
100
C57Bl/6 (n=12)
RIP3-/-Casp8-/- (n=12)
RIP3-/-Casp8+/- (n=12)
RIP3-/- (n=12)
Percent Servival
Fig. 6. Caspase-8 with RIP3 is critical for in
vivo resistance to bacterial infection. RIP3
−/−
caspase-8
−/−
dKO or WT mice were infected
s.c. with virulent Y. pestis KIM1001 (300 cfu)
for 68 h and spleens analyzed for bacterial
growth (A). Lethally irradiated mice, sub-
jected to bone marrow transplantation (BMT)
from the indicated genotypes (Band C), were
infected s.c. with 500 cfu of Y. pestis
KIM1001-EcLpxL and monitored for survival
(B), P<0.001 dKO vs. WT (log−rank test).
Spleens from moribund dKO BMT mice and
controls were analyzed for bacterial contents
(C). (D–J) Mice from BMT as above (D–F,H–J)
or regular dKO (G) were infected i.v. with
KIM1001-EcLpxL (500 cfu) for 42 h. Spleens
were homogenized and analyzed for cyto-
kines by ELISA (as cytokine/g tissue) (D–G). (H
and I) CD11b-positive myeloid cells in spleens
were analyzed for cell death with live/dead
blue and annexin V stain. (J) Liver sections
were stainedwith hematoxylin and eosin and
subjected to microscopy (400×). Foci con-
taining inflammatory cells (mostly neu-
trophils) are shown, with visible pockets
containing bacteria indicated by arrows.
Shown is a representative experiment out of
two to three performed. *P<0.05, **P<0.01
(Mann-Whitney Utest).
Weng et al. PNAS
|
May 20, 2014
|
vol. 111
|
no. 20
|
7395
IMMUNOLOGY

as previously indicated (25). Caspase-8 activity (Promega) was measured
after 2 h.
ACKNOWLEDGMENTS. We thank Kelly Army, Gail Germain, and Anna
Cerny for help with mice; Shubhendu Ghosh for assistance with the
manuscript; TeChen Tzeng for help with microscopy; Vishva Dixit
(Genentech, Inc.) for providing RIP3 KO; Douglas R. Green and Christo-
pher Dillon for sending caspase-8 RIP3 dKO mice; Joan Mecsas and Mary
O’Riordan for providing Y. pseudotuberculosis,Y. enterocolitica,and
Salmonella;D.M.S.(dmitryshay@emory.edu) for sharing cells from
previously unpublished casp8 cKO mice; and GSK: P.A.H., J.B., P.J.G. (peter.j.
gough@gsk.com), for providing RIP3 inhibitors and previously unpublished
RIP1 inhibitors. The work was supported by National Institutes of Health
(NIH) Grants AI07538 and AI057588-American Recovery and Reinvestment
Act (to E.L.), AI060025 (to N.S.), AI64349 and AI083713 (to K.A.F.), and
AI095213 (to G.I.V. and N.S.), the Norwegian Cancer Society, and the Research
Council of Norway. The study also used core services supported by University of
Massachusetts Diabetes and Endocrinology Research Center Gr ant D K325 20 and
the University of Massachusetts Core Electron MicroscopyFacility (supported
by NIH/National Center for Research Resources Award S10RR027897).
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