Review

Programmed necrosis in microbial

pathogenesis
Haripriya Sridharan and Jason W. Upton
Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, 2506 Speedway,
Stop A5000, Austin, TX 78712-1191, USA

Programmed cell death is an important facet of host

pathogen interactions. Although apoptosis has long
been implicated as the major form of programmed cell
death in host defense, the past decade has seen the
emergence of other forms of regulated death, including
programmed necrosis. While the molecular mechanisms
of programmed necrosis continue to be unveiled, an
increasing number of viral and bacterial pathogens
induce this form of death in host cells, with important
consequences for infection, control, and pathogenesis.
Moreover, pathogen strategies to manipulate or utilize
this pathway are now being discovered. In this review,
we focus on a variety of viral and bacterial pathogens
where a role for programmed necrosis is starting to be
appreciated. In particular, we focus on the mechanistic
details of how the host or the pathogen might appropriate this pathway for its own benefit.
Controlled cell death pathways
Programmed cell death (PCD) plays a significant role in the
development, immune homeostasis, and host defense of
multicellular organisms. For nearly 30 years, PCD has
been synonymous with apoptosis, and apoptosis remains
the most well studied of the PCD pathways. This controlled
form of suicide is an important arm of innate host defense
that serves to limit pathogens, and allows the immune
system to quickly and effectively dispose of dead and dying
cells.
In addition to apoptosis, it is becoming clear that other
modes of cell death are carefully controlled by eukaryotic
cells [13] (Box 1). Necrosis has been traditionally
regarded as accidental and uncontrolled cell death. However, in the past several years, this view has been challenged by clear evidence that some forms of necrotic death
are genetically programmed. Similar to apoptosis, necrosis
can also be initiated in response to various extrinsic and
intrinsic signals and carried out through specific signaling
cascades resulting in death. Such necrotic cell death is
Corresponding author: Upton, J.W. (upton@austin.utexas.edu).
Keywords: necrosis; necroptosis; RIP1; RIP3; MCMV; vaccinia; reovirus; Mycobacterium; Salmonella; Yersinia; Coxsackie B virus; cell death; innate immunity; host
defense; TNF; Listeria; Human adenovirus; influenza; EPEC; PKR; IRF3.
0966-842X/$ see front matter
2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.01.005

commonly referred to as programmed necrosis, regulated

necrosis, or necroptosis.
Programmed necrosis can be initiated by death ligands,
Toll-like receptor (TLR; see Glossary) ligands, or microbial
Glossary
Adapter proteins involved in signal transduction
Apoptosis-associated speck-like protein containing a caspase recruitment domain
(ASC): adapter protein that functions to bridge procaspase-1 and other proteins
within the inflammasomes, leading to procaspase-1 cleavage and activation.
TIR domain-containing adapter-inducing interferon-b (TRIF): adapter protein
that functions downstream of TLRs 3 and 4 to mediate downstream signals
resulting in immune responses.
Interferon (IFN) response
Interferon regulatory factor 3 (IRF3): member of the interferon regulatory factor
(IRF) family of transcription factors. Activated in response to viral nucleic acids
by phosphorylation. Important for the transcription of IFNab and IFNb, as well
as other genes important in antiviral responses.
Janus kinase (JAK): family of intracellular, nonreceptor tyrosine kinases that
mediate signal transduction initiated from IFNs. Activated by autophosphorylation and subsequently phosphorylates and activates specific STAT proteins.
Mitochondrial antiviral signaling protein (MAVS): adapter protein that transmits signals from the cytosolic RNA sensor, RIG-1, resulting in activation of IRF3
and transcription of IFNa and IFNb genes.
Signal transducers and activation of transcription (STAT): family of transcription factors that are activated by JAKs. Upon activation, homo- or heterodimerize and translocate to the nucleus to transcriptionally activate many IFNstimulated genes.
Stimulator of interferon genes (STING): adapter protein involved in host
response to cytosolic pathogen-derived or self-DNA. Acts by activating transcription factors IRF3 and STAT6.
Molecules involved in cell death pathways
Cellular FLICE-like inhibitory protein (cFLIP): potent negative regulator of caspase-8 present as long (cFLIPL) and short (cFLIPs) isoforms. cFLIPL inhibits both
apoptosis and programmed necrosis, whereas cFLIPs inhibits apoptosis but
sensitizes cells towards programmed necrosis.
Cellular inhibitor of apoptosis protein (cIAP): family of ubiquitin E3 ligases
important for regulation of apoptosis and programmed necrosis. Loss of cIAPs
sensitizes cells towards programmed necrosis by facilitating the spontaneous
assembly of an RIP1RIP3 containing signaling complex, termed the ripoptosome.
Fas-associated death domain (FADD): adapter protein that functions downstream of some death receptors (e.g., Fas and TNF) to mediate downstream
signals through the formation of a death-associated signaling complex (DISC).
Loss of FADD potentiates RIP1RIP3 programmed necrosis.
Mixed lineage kinase domain-like (MLKL): direct target of RIP3 kinase activity
and important for mediating programmed necrosis downstream of RIP3.
Phosphoglycerate mutase family member 5 (PGAM5): a mitochondria-associated phosphatase that is activated by MLKL in certain conditions of programmed necrosis. Dephosphorylates Drp1 that leads to mitochondrial fission.
Reactive oxygen species (ROS): oxygen containing byproducts of metabolism,
including oxygen anions and peroxides. Important second messengers and play
roles in signal transduction leading to inflammation and cell death.
Pattern recognition receptors (PRRs)
Nucleotide oligomerization domain (Nod)-like receptor (NLR): intracellular
PRRs that initiate immune response to injury, toxins, or microbial infection.
Activation of some NLRs can result in the assembly of inflammasomes that
activate caspase-1.
Toll-like receptor (TLR): membrane-associated PRRs that are involved in recognizing a variety of pathogen-associated molecular patterns (PAMPs) and initiating downstream signal cascades resulting in host responses during infection.

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Box 1. Forms of programmed cell death

RHIM

RIP3
RIP1

vIRA

RIP3
DAI
RHIM

RIP3
MLKL

RHIM

RHIM

RHIM

RHIM

RHIM

200

MCMV

TLR3

RHIM

infection. The most well-studied signaling pathway that is

induced by death ligands is tumor necrosis factor (TNF).
Paradoxically, it has been known for over 15 years that
TNF can induce apoptosis in some instances and necrosis
in others [4]. Unlike apoptosis, programmed necrosis is
independent of caspase activity, and inhibition or lack of
caspase-8 can sensitize cells to programmed necrosis [5].
Indeed, many additional proteins involved in regulation of
apoptosis, including Fas-associated death domain (FADD),
cellular inhibitor of apoptosis proteins (cIAPs), and cellular
FLICE-like inhibitory proteins (cFLIPs), are implicated in
regulation of programmed necrosis [2]. Signaling from
TNF receptors activates the receptor-interacting protein
(RIP) kinases 1 and 3, which then play central roles in
mediating downstream signals. Several extrinsic necrotic
signals from TNF, TLR3 activation, and certain virus
infections converge on RIP3 [6] (Figure 1). Several recent
reviews have focused on the signaling cascade from TNF
resulting in activation of the RIP1RIP3 complex termed
the necrosome [2,7]. Activation of RIP1 and RIP3 involve
auto- or cross-phosphorylation and dimerization through a
motif called a RIP homotypic interaction motif (RHIM).
Recently, it has been demonstrated that RIP1 is dispensable for some extrinsic signals (Figure 1); however, RIP3
plays an essential role in all, thus forming the crucial
convergence point for execution of necrosis. Activated
RIP3 then phosphorylates and activates downstream targets, ultimately leading to programmed necrosis. One
common downstream substrate that is activated in all
cases of RIP3-dependent necrosis is the pseudokinase
mixed lineage kinase domain-like (MLKL) [810]. MLKL
therefore probably comprises an essential step in necrosis
progression, although events downstream of MLKL are
just beginning to be elucidated. Recent studies suggest
activation results in oligomerization and plasma membrane association of MLKL, which promotes calcium

Death
receptors

RIP3
TRIF

Apoptosis: characterized by cell shrinkage, nuclear condensation,

chromosomal fragmentation, membrane blebbing, and eventual
breakdown into apoptotic bodies that are detected and disposed of
by phagocytes. Requires the activation and activity of cysteine
proteases known as caspases. Induced by external stimuli, such as
death receptor ligation or pattern recognition receptor (PRR)
activation, or by internal stimuli that influence mitochondrial
integrity. Caspases exist as latent zymogens in the cell. Initiator
caspases, such as caspases-8 and -9, are activated in response to
signals, which then activate executioner caspases, caspases-7 and 3, that target cellular substrates leading to apoptotic death.
Programmed necrosis: characterized by organelle damage, cell
swelling, and rupture of the plasma membrane, leading to the
release of cytoplasmic contents. Associated with inflammation.
Some forms of programmed necrosis are mediated by the RIP3
kinase. See text for details.
Pyroptosis: caspase-1-dependent, nonapoptotic cell death. Characterized by loss of plasma membrane integrity, release of
cytoplasmic contents, and tightly associated with the maturation
and secretion of inflammatory cytokines, including interleukin (IL)1b and IL-18. Caspase-1 is activated by the assembly of the
inflammasome, comprising activated cytosolic pathogen-sensing
NLRs [nucleotide oligomerization domain (Nod)-like receptors] that
recruit caspase-1 directly or through the adaptor ASC (apoptosisassociated speck-like protein containing a caspase recruitment
domain) [86,87].

Programmed necrosis
TRENDS in Microbiology

Figure 1. Central role of RHIM domains in programmed necrotic signaling.

Extrinsic signals from death receptors, TLRs, or viruses require RHIM containing
adapters to activate programmed necrosis. RIP3 associates with either RIP1
(TNFR1), TRIF (TLR3) or DAI (MCMV) through homotypic interactions of the RHIM
domain. This subsequently activates MLKL that then conveys the signal
downstream. vIRA protein of MCMV inhibits RIP3DAI-mediated signaling during
infection through its RHIM domain by binding and sequestering RIP3 to prevent
complex formation. Abbreviations: RHIM, RIP homotypic interaction motif; TLR,
Toll-like receptor; RIP, receptor-interacting protein; TNFR1, tumor necrosis factor
receptor 1; TRIF, TIR domain-containing adapter-inducing interferon-b; MCMV,
murine cytomegalovirus; MLKL, mixed lineage kinase domain-like; vIRA, viral
inhibitor of RIP activation.

and/or sodium influx, and ultimately leads to membrane

lysis [11,12]. However, in some cell types, MLKL activates
PGAM5, resulting in mitochondrial fission, reactive oxygen species (ROS), and necrosis [13]. It has been demonstrated that RIP3-dependent programmed necrosis does
not require mitochondria or ROS production in mouse
fibroblast and endothelial cell lines [14], suggesting that,
similar to RIP1, the requirement for mitochondria and
ROS in programmed necrosis might be specific to certain
agonists, cell types, or conditions. Thus, the minimal
requirement for execution of RIP3-dependent programmed necrosis remains an important unanswered
question.
Pyroptosis is another important form of nonapoptotic
programmed cell death (Box 1). Initially described as the
caspase-1-dependent apoptosis of macrophages during
Salmonella enterica serovar Typhimuriumn (S. Typhimurium) infection [15], further investigations revealed significant morphological and biochemical differences between
apoptosis and pyroptosis [1,16]. Although dependent upon
the activity of caspases, pyroptosis results in loss of plasma
membrane integrity and is associated with release of
inflammatory cytokines. Owing to these features, pyroptosis has multiple potent proinflammatory effects that
influence infection and immunity.
Necrosis and infection
Cellular suicide initiated by infected cells constitutes an
effective mechanism to contain pathogen replication and
spread. At the same time it facilitates the uptake and
clearance of infected cells by phagocytes of the immune

Review
system. The importance of apoptosis as a host defense is
underscored by the plethora of inhibitors encoded by different pathogens to suppress apoptosis [17,18]. Viruses
and bacteria also usurp the apoptotic pathway to aid their
replication as well as to avoid detection and clearance by
immune cells. In some cases, apoptosis positively correlates with disease progression and pathology. One example
is HIV-1, which induces bystander apoptosis in CD4+ T
cells [19]. This depletes the cellular reservoir that mounts
an immune response against the virus, ultimately leading
to increased viral titers and AIDS. Similarly, the bacterium Mycobacterium avium induces apoptosis in infected
macrophages, and although this may lead to effective
clearance of the bacterium [20], other studies suggest
apoptosis can promote M. avium spread [21], highlighting
the intricate dance between life and death. Regardless,
apoptosis is clearly an important facet of the host
pathogen interaction that is used by both sides.
Recently, we have begun to appreciate an analogous role
for necrosis in the hostpathogen interplay. In some cases,
programmed necrosis is clearly a host defense mechanism
against infection. In others, it is coopted by intracellular
bacteria and viruses to aid in dissemination and spread. In
all these cases, necrosis clearly influences the virulence
and pathogenesis of the infectious disease. Here, we will
discuss a number of viral and bacterial pathogens and the
influence programmed necrosis has on their infection.
Vaccinia virus
Accumulating evidence clearly indicates that programmed
necrosis, at least in some cases, is a critical antiviral host
defense mechanism. One such case is infection by vaccinia
virus (VV). This large, double-stranded DNA virus encodes
a plethora of immune and cell modulatory genes to facilitate infection, among them B13R, an ortholog of the wellcharacterized cowpox serpin, CrmA. Similar to CrmA,
B13R can bind and inhibit caspases, including caspase-8
[22,23]. B13R is necessary for VV pathogenesis in mice
[24], presumably to prevent apoptosis of infected cells.
However, VV infection of mouse fibroblasts results in
the sensitization of these cells to TNF-induced, caspaseindependent necrotic cell death requiring B13R [25]. This
apparent paradox was afforded mechanistic explanation
when it was shown that RIP1 participates in death receptor-induced necrosis [26] as well as death of VV infected
cells [27]. Thus, inhibition of apoptosis, at least in some
cases, promotes TNF-dependent programmed necrosis.
The RIP1RIP3 necrosome was clearly implicated as an
important mediator of host defense in VV infected mice,
because genetic ablation of RIP3 and TNF receptor 2
(TNFR2) ameliorated extensive necrotic damage and
inflammation [27,28]. RIP3 / mice showed reduced
pathology, but suffered significantly higher viral titers,
and succumbed to infection. Conversely, wild type mice
controlled VV infection, but were afflicted with more severe
tissue pathology [28]. Taken together, these data indicate
that VV is controlled by the host through TNF-induced,
RIP1RIP3-dependent programmed necrosis. Although
this antiviral strategy results in enhanced pathology associated with VV infection, it limits replication of the virus to
protect from infection.

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Murine cytomegalovirus
Another virus demonstrating an antiviral role for necrosis
is murine cytomegalovirus (MCMV). MCMV is a herpesvirus and establishes a life-long infection of its host. As
such, the fate of infected cells is a critical issue that MCMV
must manipulate to facilitate replication and persistence.
Similar to other herpesviruses, MCMV encodes a number
of inhibitors of apoptosis [29], including a viral inhibitor of
caspase activation (vICA) that directly targets caspase-8
[30]. Although MCMV and VV inhibit caspase-8 during
infection, the outcomes of infection by these viruses are
very different. Unlike VV, wild type MCMV infection does
not sensitize cells to TNF-induced cell death [31]. This is
due in large part to the MCMV M45 gene product, which
encodes the viral inhibitor of RIP activation (vIRA). vIRA is
a RHIM-containing protein that specifically binds and
inhibits RIP3 (Figure 1) [32,33]. A recombinant MCMV
encoding vIRA with a mutated RHIM (MCMVmutRHIM)
was severely attenuated in both immune-compromised
and normal hosts, but was completely rescued in RIP3 /
mice [33]. These results unequivocally demonstrated that
inhibition of RIP3 RHIM-dependent programmed necrosis
by vIRA is critical for MCMV infection.
Unlike VV, death receptors and RIP1 were not involved in
MCMVmutRHIM-induced programmed necrosis. Instead, it
requires the DNA-dependent activator of interferon regulatory factors (DAI/ZBP1) (Figure 2), a RHIM-containing pathogen recognition receptor involved in the innate immune
response to DNA virus infection [3436]. Expression of DAI
sensitizes cells to MCMVmutRHIM-induced necrosis, and a
RIP3DAI interaction is detectable in MCMVmutRHIM virus
infected cells. Similar to loss of RIP3, RNAi mediated knockdown or genetic ablation of DAI significantly reduces necrosis
and rescues MCMVmutRHIM virus pathogenesis in vitro and
in vivo. Together, these genetic and biochemical data indicate
that programmed necrosis is a potent antiviral strategy
employed by the host to combat viral infections, such as
VV and MCMV infection, and some viruses have developed
elegant strategies to circumvent this defense mechanism.
Reovirus
Mammalian reovirus is a nonenveloped double-stranded
RNA (dsRNA) virus long known to induce cell death during
infection both in vitro and in vivo (reviewed in [37,38]).
Reovirus-induced cell death is mediated by death receptor
signaling, particularly via the death cytokine TNF-related
apoptosis-inducing ligand (TRAIL) [39]. Although reovirus-induced programmed cell death appears to play little
role during infections in vitro, infection of target tissues in
vivo, including the heart and central nervous system,
results in extensive reovirus strain-specific tissue injury
due to PCD. In some cases, reovirus induction of cell death
is necessary for maximal virus growth in vitro and in vivo,
suggesting that reovirus has coopted this host defense
mechanism to facilitate its own pathogenesis [37]. Moreover, cell death is also strain-dependent, with the Type 3
(T3) strains inducing more death than other laboratory
strains, such as Type I (T1) [40]. T3 strains of reovirus
cause fatal encephalitis in neonatal mice that is dependent
upon the activity of proapoptotic Bid, highlighting the
important contribution of apoptosis to the pathology of
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Coxsackie B virus
Pore-forming toxins
Vaccinia
Reovirus
IAV
Mycobacteria
EPEC

Ca2+
peturbaon

DR

Calpains

Salmonella
RIP1
Yersinia
C. perfringens toxin

RIP3

Programmed necrosis

IRF3

Adenovirus
Listeria

DAI
MCMV

TRENDS in Microbiology

Figure 2. Multiple routes to programmed necrosis during infection. Viral and bacterial pathogens, including vaccinia, reovirus, IAV, Mycobacterium, and EPEC, activate the
RIP3-dependent necrotic pathway via death receptors (DRs), whereas Salmonella, Yersinia, and the Clostridium perfringens toxin induce necrosis dependent on RIP1 kinase
activity. MCMV elicits and inhibits an analogous DAI-dependent pathway leading to RIP3. Other pathogens induce programmed necrosis through IRF3 or by changes in
intracellular calcium via mechanisms that are not currently understood. ? indicates an unknown connection. Abbreviations: IAV, influenza A virus; EPEC, enteropathogenic
Escherichia coli; RIP, receptor-interacting protein; MCMV, murine cytomegalovirus.

disease [41]. Correspondingly, the T3 strains also induce

more effector caspase and nuclear factor-kB (NF-kB) activation. Surprisingly, neither was required for cell death in
murine L929 cells, and T3-induced death was reduced by
the RIP1 inhibitor, necrostatin-1 (nec-1). This suggests
that the predominant form of death in L929 cells is RIPdependent programmed necrosis rather than apoptosis
(Figure 2) [42]. The initiating events for necrosis during
reovirus infection may be different than those inducing
apoptosis, because necrosis requires viral RNA and protein
synthesis, unlike apoptosis where viral penetration is
sufficient [4345]. It is conceivable that both these forms
of cell death contribute to reovirus infection, but may
differentially mediate damage in specific tissues or cell
types. Although reovirus-induced apoptosis has a positive
effect on viral replication, it remains to be determined
whether necrosis plays a significant role in virus growth
and spread. In either case, it will be important to determine the contribution of programmed necrosis in the context of in vivo infection and assess its role in pathogenesis
alongside apoptosis.
Influenza A virus
Programmed cell death plays an important role in influenza A virus (IAV) infections. IAV inhibits apoptosis early
during infection, through the action of its nonstructural
protein 1, but actively induces apoptosis at later stages of
infection [46]. A recent study has identified cIAP2 as a
critical inhibitor of programmed necrosis necessary to
maintain tissue homeostasis during IAV infections [47].
Unlike wild type mice, which eventually recovered from
infection, cIAP2-deficient mice succumbed to infection due
to massive necrosis and hemorrhaging of the airway passage epithelia. Programmed necrosis was mediated by
the death cytokine FasL and required RIP1 and RIP3.
202

Chemical inhibition of RIP1 or genetic depletion of RIP3

rescued infected cIAP2 / mice from tissue damage and
improved animal survival. Interestingly, programmed
necrosis triggered by IAV infection in cIAP2 knockout mice
did not affect viral titers, immune cell activation, or the
inflammatory cytokine response [47]. This suggests that
programmed necrosis in the case of IAV infection does not
act as a host defense or even as a viral strategy employed
by IAV to complete its life cycle. Instead, it is clear evidence
that necrosis contributes to the pathology and severity of
IAV-induced disease, thereby having important consequences for patient outcome, especially in patients where
cIAP2 function may be compromised.
Mycobacterium
The past few years have seen an increasing number of
studies identifying programmed necrosis as a consequence
of bacterial infections. Mycobacterium tuberculosis (MTB)
infection proceeds in several distinct but inter-related
stages (reviewed in [48,49]). The bacteria invade alveolar
macrophages in the lung, leading to robust localized
inflammation, with the release of inflammatory cytokines,
such as TNF, which promote granuloma formation
(reviewed in [49]). However, the relationship among
MTB, TNF, and programmed necrosis is complex.
Although TNF plays an important role in control of
MTB, excess can cause tissue damage and organ dysfunction. Emphasizing this delicate balance, a series of recent
studies elegantly demonstrated that both excess and insufficient TNF exacerbate outcomes of TB infection through
programmed necrosis [5052]. Excess TNF during infection causes an RIP1/RIP3-dependent increase in ROS that
initially aids in intracellular control of the bacteria in
macrophages. However, it ultimately leads to TNFmediated, RIP1RIP3-dependent programmed necrosis

Review
and release of MTB [50]. Thus, initial protection afforded
the host in control of intracellular bacteria by ROS is
negated by host cell necrosis and release of the bacteria
into the extracellular compartment. Chemical or RNAimediated inhibition of programmed necrosis by targeting
RIP1, RIP3, MLKL, or PGAM5 prevented both the initial
bactericidal activity as well as subsequent necrosis of
macrophages, resulting in enhanced extracellular bacterial loads, which has an adverse outcome for the host.
However, targeting the pathway downstream of ROS production, but upstream of cell lysis, through blockade of
cyclophilin D or acid sphingomyelinase rendered the host
hyper-resistant to infection. By maintaining a low intracellular bacterial burden, but also preventing macrophage
necrosis and extracellular dissemination, the infection is
efficiently controlled. Although these studies were performed in a zebrafish model system, they highlight the
tight balance between infection, death-promoting cytokines, and programmed necrosis, as well as how targeting
the necrotic pathway at precise points could potentially
improve patient outcome in tuberculosis therapy.
Salmonella
S. Typhimurium is a highly virulent intracellular bacterial
pathogen. S. Typhimurium infection of normal mice results
in rapid mortality coincident with excessive necrotic death
of infected macrophages. This is dependent upon type I
interferons (IFNs) and mediated by the interferon receptor
1 (IFNAR1) [53]. Macrophages from IFNAR / mice displayed better survival and were therefore better able to
contain bacterial burdens earlier during infection, as compared with those from wild type mice. Interestingly, macrophages treated with nec-1, or those derived from RIP3 /
mice, were protected from death, confirming a role for RIP1
RIP3 programmed necrosis during S. Typhimurium infection. However, it remains unclear how programmed necrosis
contributes to the pathogenesis of S. Typhimurium, because
RIP3 / mice showed only modest improvements in survival
compared with wild type animals [53]. Inhibition of caspase1 activity also modestly reduced the lethality in infected
macrophages, suggesting that multiple pathways contribute to lethality of the host during S. Typhimurium infection. Nevertheless, RIP1RIP3-dependent death influences
survival of infected macrophages during S. Typhimurium
infection in vitro and in vivo (Figure 2). RIP1 was shown to
associate with IFNAR1 [53], but the actual mechanism of
necrosome activation in this scenario is not understood. This
warrants additional work to understand the physiological
role of programmed necrosis during infection of this important bacterial pathogen.
Yersinia
Yersinia outer membrane proteins (YOPs) are secreted
from different Yersinia strains, contributing to virulence
and pathogenesis of the bacteria by counteracting host
innate and adaptive immunity [54]. In macrophages, the
Yersinia enterocolitica YopP (YopJ in Yersinia pestis)
induces caspase-8 activation and apoptotic death. However,
in dendritic cells (DCs), YopP induces death simultaneously
through both apoptosis and necrosis [55]. Treatment of
Yersinia infected DCs with caspase inhibitors partially

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protected cells from death, suggesting that both caspasedependent and -independent pathways of death operate in
DCs. Injection of YopP into DCs induces formation of a
caspase-8FADDRIP1 complex independent of death
receptor activation [56]. This is reminiscent of the complex
termed the ripoptosome, which is induced by the depletion
of cIAPs or DNA damaging agents [57,58]. YopP is a cysteine
protease whose activity is essential for cleavage of caspase-8
and RIP1 [56]. Cleaved RIP1 amplifies the apoptotic
response [59,60]; how it acts as a pronecrotic mediator
remains to be determined. Chemical depletion of RIP1 in
this system by geldanamycin implicates RIP1 as a necessary
mediator of YopP death. By contrast, YopJ-induced necrosis
in infected macrophages was unaffected by RIP1 kinase
inhibition with nec-1 [61]. These apparently conflicting
results could be reconciled by the finding that geldanamycin
also depletes RIP3 levels [28]. Thus, the necrosis induced by
both YopP in DCs and YopJ in macrophages may be RIP3dependent, but RIP1-independent (Figure 2). However, a
definitive role for RIP3 in Yop-mediated necrosis, as well as
how YopP/J-induced cell death influences Yersinia pathogenesis remain open questions.
Enteropathogenic Escherichia coli (EPEC)
Many bacterial gene products, such as the Yops discussed
above or pore-forming toxins discussed later, induce eukaryotic cell death. However, recently the type III effector
protein of pathogenic E. coli, NleB, was identified as an
inhibitor of cell death signaling [62]. NleB has a novel Nglucosamineacetyl (NGlnAc) transferase activity that targets a conserved arginine in the death domains of TRADD,
FADD, TNFR1, and RIP1, effectively inhibiting the association of the death domains to abolish downstream signaling in response to death cytokines. Moreover, the
NGlnAc activity also suppressed TNF-induced programmed necrosis in RIP3 overexpressing HeLa or
HT29 cells. In mice infected with NleB-deleted strains,
the bacteria were unable to effectively colonize the host,
showing reduced colony forming units in stool and colon, as
compared with mice infected with wild type strains [62],
highlighting the importance of these pathways as protective to the host and helping to clear infection. Further
investigations into the mechanisms of host cell death during in vivo infection will probably yield important insights
into the interplay between E. coli and its host.
Interferon-dependent, noncanonical pathways for
necrosis
The intracellular bacterium Listeria monocytogenes and
human adenovirus (hAdv) both induce a form of programmed necrosis with rapid kinetics in infected macrophages [63]. Intriguingly, this cell death was mediated by
the transcription factor, IRF3 (Figure 2). RIP3 appeared to
play no role in this form of programmed necrosis, at least
during hAdv infection. Although insight into the mechanism of IRF3-induced necrosis is lacking, no difference in
cytokine profiles between wild type and IRF3 / mice were
detected, indicating that transcriptional activity of IRF3 is
probably not involved. Neither Listeria, nor HAdv,
mutants unable to escape from the endosome into the
cytosol induced IRF3-dependent necrosis. Surprisingly,
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death was independent of upstream sensors such as
STING, MAVS, and DAI [63]. The bacterial burden in
Listeria-infected wild type mice was doubled in comparison
to IRF3 / mice, suggesting that Listeria uses programmed necrosis to aid bacterial dissemination. However, this trend was inversed for hAdv because viral
burdens were higher in the livers of IRF3 / infected mice
as compared with wild type. This serves as yet another
example of a necrotic pathway used as both a host defense
and pathogen survival strategy.
Recently, another study established the role of the protein kinase R (PKR) in programmed necrosis mediated by
IFNs [64]. PKR has been previously implicated in apoptosis
caused by various stimuli, including dsRNA, lipopolysaccharide (LPS), and TNF [65,66]. IFN-mediated necrosis
required JAK/STAT signaling, PKR, RIP1, and RIP3, and
although primarily demonstrated with IFNg, was similar
for both type I and type II IFNs [64]. Interestingly, the
kinase activity of RIP1 was dispensable; instead, IFN
induced PKR associated with RIP1 to form the necrosome
and initiate programmed necrosis. Moreover, the elucidation of this IFN-dependent pathway calls into question
whether type II IFN and PKR could also be involved in S.
Typhimurium-induced programmed necrosis [53].
Viruses have been shown to inhibit nearly every step of
IFN production and signaling, including activation of
IRF3, JAK/STAT signaling, and PKR [67]. In addition to
inhibiting the antiviral functions of IRFs and IFNs, these
viral proteins could potentially play an unappreciated role
in inhibiting host cell death. This could explain why IFNmediated programmed necrosis is not a more commonly
observed phenomenon with virus infections. Future investigations into the effect of these pathways on programmed
necrosis will probably be illuminating.
Cell death induced by pore-forming toxins
Many bacteria encode pore-forming toxins that are crucial
to pathogenesis, contributing to sepsis and tissue damage
in the host. In some cases, the same toxin can initiate
different host cell death pathways in a dose-dependent
manner [68]. b-Toxin from Clostridium perfringens [69],
a-toxin from Clostridium septicum [70,71], and Helicobacter pylori VacA [72] all cause a primarily necrotic death. In
the case of C. perfringens, necrosis is inhibited by nec-1,
suggesting a role for RIP1 kinase activity (Figure 2). However, in most other systems, it remains to be determined
whether RIP1 and/or RIP3 play any role in executing toxininduced death. The inflammasome is activated by many
pore-forming toxins [73,74]. Recent evidence indicates that
RIP3 can directly activate the NLRP3 inflammasome independent of its necrotic signaling [75,76], suggesting the
possibility that both pyroptosis and programmed necrosis
actively contribute to death by soluble toxins. Inflammation has generally been considered a secondary effect of
necrosis due to release of danger-associated molecular
patterns (DAMPs) from the necrotic cell. However, inhibition of cIAPs, important regulators of programmed necrosis, resulted in increased RIP3-dependent activation of the
inflammasome [75]. Other components recruited to the
necrosome, including MLKL and PGAM5, have also been
implicated [76], suggesting a complex interplay among cell
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death modalities, inflammatory signaling, and bacterial

toxins.
Calcium, calpains, and necrosis
Many bacterial toxins and some viruses, such as Coxsackie
B virus (CVB), induce necrotic cell death by activating
calpains (Figure 2), a family of calcium-dependent proteases known to promote cell death. CVB is an enterovirus
that depends on cell lysis for virus release at the end of its
life cycle. Many studies conducted on nonpolarized cells
identified activation of caspases and a role for apoptosis in
CVB release [7779]. However, in polarized cells, CVBinduced cell death was inhibited by a calpain inhibitor or
knock-down of calpain-2, but not caspase inhibitors [80].
Importantly, knock-down of calpain-2 by RNAi significantly inhibited CVB release from cells, pointing to an
important role for calpain-mediated necrosis in the life
cycle of CVB. The role of the RIP kinases in CVB-induced
necrosis is not understood. Many forms of necrosis exhibit
altered intracellular calcium levels, calpain activation, and
lysosomal degradation [7,81]. However, the connection
between RIP3 activation and increases in calcium-dependent signaling is not entirely clear. It has been suggested
that signals from calcium perturbations do not require
RIP1, RIP3, or MLKL to induce programmed necrosis,
but is dependent on PGAM5 [13]. This could be contextspecific depending on the cell type and/or necrotic stimuli.
A more detailed look into possible connections between
RIP3 activation and effects of calcium is needed to better
understand this regulation.
Other potential inhibitors of necrosis
So far, only two proteins from pathogens, namely vIRA
from MCMV and NleB from E. coli, have been shown to
directly interact with and inhibit programmed necrosis to
influence an infection in a host. Viruses of the poxvirus and
herpesvirus families encode homologs of cFLIP, which are
strong apoptosis inhibitors [2]. Intriguingly, a subset of
these vFLIP molecules can also inhibit programmed necrosis [27], although they have not been tested in a natural
infection model. However, as the role of necrosis in viral
and bacterial infection continues to expand, it is probable
that other inhibitors of necrosis will be identified. Viruses
that encode caspase-8 inhibitors [2] could also encode for as
yet undiscovered inhibitors of programmed necrosis. The
ribonucleotide reductase large subunits of herpes simplex
virus (HSV)-1 and HSV-2, orthologs of MCMV vIRA, protect from cell death in isolation [82], although further
investigation is necessary to assess any potential role in
suppressing programmed necrosis. It is tempting to speculate that inhibition of necrosis may be a common strategy
employed by many more pathogens to evade the host
immune system.
Concluding remarks
Data from studies of several pathogens have indicated an
important role for programmed necrosis in mediating
virulence and pathogenesis of infectious disease. As it
becomes appreciated that necrosis is not an accidental
death modality, the studies discussed in this review highlight the emerging concept of how infections by a variety of

Review

Trends in Microbiology April 2014, Vol. 22, No. 4

Table 1. Summary of microbial induction and inhibition of programmed necrosis

Pathogen

Vaccinia virus
MCMV
Reovirus
Influenza A virus
Mycobacterium
Salmonella
Yersinia
Enteropathogenic
Escherichia coli
Listeria monocytogenes
Human adenovirus
Clostridium perfringens
Clostridium septicum
Helicobacter pylori
Coxsackie B virus

Mechanism
of programmed
necrosis
TNFRIP1RIP3
DAIRIP3
DRRIP1
FasL (TRAIL)RIP1RIP3
TNFRIP1RIP3

Pathogen influence
on programmed
necrosis
Promotes
Inhibits
Promotes
Promotes
Promotes

Pathogen-associated
programmed necrosis
inhibitorb or inducer c
ND
vIRA(M45) b
ND
ND
ND

Refs

ND
YopJ c
NleB b

Consequence of
programmed necrosis
during infection
Host defense
Host defense
ND
Enhanced pathology
Benefit to both host
and pathogen d
Limits host defense
ND
Host defense

IFNRIP1RIP3
RIP1RIP3
DRRIP1RIP3

Promotes
Promotes
Inhibits

IRF3
IRF3
RIP1, Calpains
Calpains
ND a
Calpain-2

Promotes
Promotes
Promotes
Promotes
Promotes
Promotes

ND
ND
B toxin c
A toxin c
Vac A c
ND

Promote dissemination
Host defense
ND
ND
ND
Promote dissemination

[60]
[60]
[66]
[67,68]
[69]
[77]

[2224]
[29,30,33]
[39]
[44]
[47]
[50]
[52,53,58]
[59]

ND, not determined.

Pathogen-associated programmed necrosis inhibitor.

Pathogen-associated programmed necrosis inducer.

TNF-mediated signaling and ROS facilitates early control of intracellular bacteria, but also promotes cell lysis and bacterial dissemination. See text for details.

pathogens actively engage signal transduction pathways

leading to necrosis of the host cell. In some cases, programmed necrosis functions as an antiviral/antibacterial
response against infection; in others, it is utilized by the
pathogen to aid its dissemination (Table 1). More importantly, necrosis can contribute to inflammation, tissue
destruction, and, therefore, disease severity and should
be considered as a component of treatment options. A
common theme that emerges from most of the above
studies is that the type of death induced by an infection
is contextual, dependent upon a multiplicity of factors,
ranging from pathogen genetics to host immune responses,
and deciphering these contexts in terms of pathogen infection should remain a top priority.
Many interesting questions remain to be answered
regarding programmed necrosis and infections (Box 2).
In addition to caspase-8 and FADD [5,83], there has been
some evidence that caspase-9 has a role in inhibition of
necrosis during MTB infection [84]. Interestingly, a genome-wide screen for genes involved in programmed necrosis identified the Bcl-2 family member Bmf [85]. It remains
to be seen whether additional apoptotic checkpoints serve
to regulate programmed necrosis. Another important question is how metabolic and chemical conditions in the host
cell determine regulation of programmed necrosis. Currently, we lack clear understanding of how intrinsic signals such as calcium flux or ROS are related to RIP kinase
signaling and the initiation of programmed necrosis, and

Box 2. Outstanding questions

 What points in the apoptotic pathway in addition to caspase-8 and
FADD serve as checkpoints for programmed necrosis?
 How are changes in ROS or calcium flux related to RIP3 activity?
 What are the mechanisms by which pathogens other than MCMV
and EPEC modulate programmed necrosis?

whether pathogen-induced changes in those conditions

represent a cause or effect of these pathways. A third
question is how the pathogens described here, and those
not discussed, engage the pathways leading to programmed necrosis. Although pathogens such as VV,
MCMV, and EPEC have been clearly linked mechanistically to programmed necrosis, it remains an open question
as to how, and even whether, other pathogens initiate,
inhibit, or otherwise manipulate this emerging form of host
defense.
Necrosis is no longer an accident. The term now encompasses an important, genetically controlled host cell
defense mechanism: one important enough for pathogens
to inhibit. This is an exciting time in the field of programmed necrosis as we start to not only understand
the molecular mechanisms mediating this form of cell
death but also how it functions in the context of human
health and infectious disease.
Acknowledgments
We sincerely apologize to the many colleagues whose work could not be
discussed or referenced owing to space limitations. We would like to
thank Jamie Waggoner for graphic design and members of the Upton
laboratory for helpful discussions and critically reading the manuscript.
Work in the Upton laboratory is supported by the Cancer Prevention and
Research Institute of Texas (CPRIT) Scholar award R1202 (J.W.U.).

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