Cell Host & Microbe

Article

Cellular Inhibitor of Apoptosis Protein cIAP2Protects against Pulmonary Tissue Necrosis duringInfluenza Virus Infection to Promote Host SurvivalIan Gael Rodrigue-Gervais,1,6 Katherine Labbe,2,6 Maryse Dagenais,3 Jeremy Dupaul-Chicoine,3 Claudia Champagne,1

Alexandre Morizot,1 Alexander Skeldon,3 Erik L. Brincks,5 Silvia M. Vidal,4 Thomas S. Griffith,5 and Maya Saleh1,2,3,*1Department of Medicine2Department of Microbiology and Immunology3Department of Biochemistry4Department of Human Genetics

McGill University, Montreal, QC H3A 1B1, Canada5Department of Urology, Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA6These authors contributed equally to this work

*Correspondence: maya.saleh@mcgill.ca

http://dx.doi.org/10.1016/j.chom.2013.12.003

SUMMARY

Cellular inhibitors of apoptosis proteins (cIAPs) areessential regulators of cell death and immunity. Thecorresponding contributions of IAPs to infectiousdisease outcomes are relatively unexplored. Wefind that mice deficient in cIAP2 exhibit increasedsusceptibility and mortality to influenza A virus infec-tion. The lethality was not due to impaired antiviralimmune functions, but rather because of death-receptor-induced programmed necrosis of airwayepithelial cells that led to severe bronchiole epithelialdegeneration, despite control of viral replication.Pharmacological inhibition of RIPK1 or genetic dele-tion of Ripk3, both kinases involved in programmednecrosis, rescued cIAP2-deficient mice from influ-enza-induced lethality. Genetic deletion of thedeath receptor agonists Fas ligand or TRAIL fromthe hematopoietic compartment also reversed thesusceptibility of cIAP2-deficient mice. Thus, cIAP2-dependent antagonism of RIPK3-mediated pro-grammed necrosis critically protects the host frominfluenza infection through maintenance of pulmo-nary tissue homeostasis rather than through path-ogen control by the immune system.

INTRODUCTION

Infection with influenza A virus causes an acute, highly conta-

gious respiratory illness that triggers cell death of infected cells

and airway epithelial destruction. Cell death is an integral part

of the immune response to influenza (Yatim and Albert, 2011).

For example, apoptosis is thought to clear infected cells to limit

viral propagation and to control runaway immunopathological

responses. However, inappropriate cell death can disrupt the

architecture and integrity of the bronchoalveolar network,

leading to barrier disruption and compromised arterial blood

Cell H

oxygenation following lethal influenza infection in the mouse

model (Sanders et al., 2013). In humans, both necrosis and

apoptosis of distal pulmonary epithelia were described as a

classical feature of H5N1 or H1N1 influenza-induced acute res-

piratory distress syndrome (Korteweg and Gu, 2008; Mauad

et al., 2010). Viruses can trigger not only apoptosis but also an

alternative form of caspase-independent cell death called

programmed necrosis (or necroptosis). Necroptosis, which

depends on the kinase activity of the receptor-interacting

proteins (RIPK)1 and RIPK3 (He et al., 2009), is emerging as a

backup host defense mechanism that restricts virus persistence

in instances when apoptosis is disabled by the virus, as exempli-

fied by vaccinia virus or the murine cytomegalovirus (Cho et al.,

2009; Upton et al., 2010, 2012). Whether necroptosis plays a

similar role in host resistance to RNA viruses is not clear (Berger

and Danthi, 2013). While cell death is effective in curbing viral

spread by removing intracellular replication niches, it places a

cost on the host by exacerbating tissue injury following infection

and, as such, must be tightly controlled.

Apoptosis and necroptosis are complementary cell death

pathways controlled by common signaling adaptors, kinases,

caspases, and ubiquitin ligases. Of these, the cellular inhibitors

of apoptosis proteins (cIAPs), through their E3 ubiquitin ligase

activity, are critical upstream regulators of both forms of cell

death (Vanlangenakker et al., 2011b). However, the exact con-

tribution of individual IAP in antagonizing cell death in vivo

has been difficult to ascertain because of their apparent

functional redundancy in mammals. Indeed, Birc2(cIAP1)�/�,Birc3(cIAP2)�/� and Birc4(XIAP)�/� mice are all viable and

display limited cell-death-related phenotypes in opposition to

Birc2�/�Birc4�/� orBirc2�/�Birc3�/�mice that die by embryonic

day E10.5 (Moulin et al., 2012). This lethality was delayed to

some extent by codeletion of Ripk1 or Ripk3, highlighting that

cIAPs function in vivo as arbiters of cell fate through repression

of RIP kinase activity. Mechanistically, cIAP1 and cIAP2 mediate

the ubiquitination of RIPK1, which prevents the formation of a

large �2 MDa RIPK1-dependent platform, named the ‘‘ripopto-

some’’ (also referred to as necrosome) (Feoktistova et al.,

2011; Tenev et al., 2011). In response to different stressors

such as viral infections or engagement of death receptors

ost & Microbe 15, 23–35, January 15, 2014 ª2014 Elsevier Inc. 23

Cell Host & Microbe

cIAP2 Maintains Lung Homeostasis during Infection

(DRs), the recruitment of high levels of RIPK3 to the necrosome

leads to caspase-independent necrotic cell death. The cIAPs

have also been shown to exert key functions in the regulation

of innate immunity. Notably, they are required for cytokine and

chemokine production by NOD1 and NOD2 (Bertrand et al.,

2009), mediate MAPK activation by TLR4 (Tseng et al., 2010),

enhance caspase-1 activation by the inflammasome (Labbe

et al., 2011), and carry out ubiquitination steps downstream of

RIG-I signaling (Mao et al., 2010). Although cIAPs are recognized

as effectors in cell death and immune signaling pathways, there

is an incomplete understanding of which of these two defense

processes are principally regulated by cIAPs during infectious

disease, and even less is known of their involvement in protec-

tion against viruses.

Here we report that deletion of cIAP2 leads to marked loss of

lung epithelium and enhanced susceptibility to influenza-virus-

induced acute lung injury. This is not a result of any defects in

antiviral immunity. Rather, cIAP2 acts to calibrate the activation

threshold of necroptosis in pulmonary epithelial cells at the level

of its regulation of the stoichiometry of FLIP isoforms and the

formation of a RIPK3 necrosome complex that is assembled in

response to DR signaling during infection.

RESULTS

Enhanced Susceptibility to Influenza Virus Infection incIAP2-Deficient Mice despite Virus ControlTo investigate whether cIAP2 influences immunity, inflammation,

and/or pulmonary tissue homeostasis to viral infections, wild-

type (WT) and Birc3�/� mice were infected intranasally with a

sublethal dose of H1N1 influenza A virus Puerto Rico/8/34 strain

(referred to as PR/8) and monitored for pathologic and virologic

parameters. After exposure to PR/8, over 75%ofWTmice recov-

ered by day 13 postinfection (p.i.) (Figure 1A), despite a loss of

25% of their original body weight (Figure 1B). In contrast,

Birc3�/� mice had increased mortality (Figure 1A; p % 0.0001)

and considerably greater weight loss (Figure 1B), despite both

genotypes not exhibiting differences in viral replication (Fig-

ure 1C) or viral titers in the lung (Figure 1D). To determine the

cellular compartment in which cIAP2 expression was required

for host protection, we generated bone marrow chimeras in

which only the hematopoietic (WT/Birc3�/�) or radio-resistantcompartment (Birc3�/�/WT) expressed cIAP2. The failure of

WT marrow to rescue Birc3�/� mice from PR/8-induced lethality

suggested that cIAP2 is primarily required in the radio-resistant

compartment (Figure 1E). Notwithstanding the ubiquitous role

of the cIAPs in immune signaling, the lethality of Birc3�/� mice

after PR/8 infection was not linked to appreciable defects in

inflammation or the innate and adaptive immune responses (Fig-

ure S1 available online; related to Figure 1). With the exception of

CXCL1, which was moderately higher in Birc3�/� mice (Fig-

ure S1A; p < 0.05), whole-lung levels of IL-1b, IL-6, CCL2, and

IFN-lwere similar between genotypes on all daysmeasured (Fig-

ure S1A). Secretion of these cytokines into the bronchoalveolar

lavage fluid (BALF) on day 3 p.i. was also not different between

genotypes (Figure S1B). Correspondingly, there was similar

recruitment of leukocytes into the lung on days 3 and 7 p.i. (Fig-

ure S1C) and the frequencies of macrophages, neutrophils, NK

cells, B cells, and T cells in the infected lung were not different

24 Cell Host & Microbe 15, 23–35, January 15, 2014 ª2014 Elsevier I

(Figure S1D). Secretion of IFN-b and CXCL10 (two antiviral de-

fense genes) by BMDCs from Birc3�/� mice was equivalent to

that of WT BMDCs when infected in vitro with Sendai virus or

transfected with 50 triphosphate dsRNA (Figures S1E and S1F),

indicating that there is no intrinsic defect in RIG-I signaling in

the absence of cIAP2. Indeed, secreted levels of IFN-a and

CXCL10 into the BALF of WT and Birc3�/� mice were similar

between genotypes (Figures 1F and 1G). Consistent with intact

antiviral immune responses capable of controlling PR/8 infection,

the total number of influenza DbNP366-specific CD8+ T cells pre-

sent in the BALF, lung parenchyma and spleen of Birc3�/� mice

was similar to that of WT mice (Figure S1G–S1J). Additionally,

influenza-specific CD8+ T cells from Birc3�/� mice were as

capable of producing IFN-g as those ofWTmice upon ex vivo re-

stimulationwith influenza peptides (Figure S1K). Finally, although

influenza-specific IgG antibody levels were reduced in the sera of

Birc3�/� mice on days 7 and 11 p.i. (Figure S1L), this decrease

did not appear to influence viral clearance in this genotype, as

the lung viral titers from the same animals were not increased

compared to WT mice (Figures 1D and 2E).

Impaired Airway Integrity Following Influenza Infectionin Birc3–/– MiceHistopathological examination of lung sections from naive mice

(Figure 2A), compared to those from infected WT mice on days

7 and 11 p.i. (Figure 2B), revealed peribronchiolar inflammation

and epithelial hyperplasia characteristic of ongoing tissue dam-

age and repair responses during PR/8 infection (Monticelli et al.,

2011). In contrast, lungs from infected Birc3�/� mice had large

regions of hemorrhage, as well as more pronounced bronchiolar

epithelial damage with extensive sloughing of the epithelial lining

(Figure 2B) and, in many cases, obstruction of the airway by

exudate and dead cells in the bronchiolar lumen. These histo-

pathological features were unlikely to be due to preexisting

differences in the airway epithelium of Birc3�/� mice, as their

bronchioles prior to infection had normal epithelial cell lining

similar to naive WT mice (Figure 2A). The observed changes to

the airway architecture of Birc3�/� mice following infection

were linked to impaired epithelial integrity by day 11 p.i.

compared to WT mice, as measured by increased translocation

into the BALF of intravenously injected BSA-fluorescein isothio-

cyanate (FITC) (Figure 2C). Probing for the epithelial cell marker

CD326 confirmed that a fraction of the cells shed into the bron-

chiolar lumen were of epithelial origin and that this phenomenon

was generally confined to Birc3�/� mice (Figure 2D). Moreover,

there was no significant difference in the pathogen burden, as

measured by immunofluorescence staining for PR/8 antigens

(Figure 2E), in the samemice that showed loss of epithelial integ-

rity (Figures 2B and 2C), reinforcing the idea that lethality and

tissue damage are independent of viral control. Collectively,

these observations indicate a role for cIAP2 in maintaining tissue

homeostasis in the lung following acute viral infection.

Caspase-Independent Cell Death of Birc3–/– AirwayEpithelial CellsBecause histological analysis of hematoxylin and eosin

(H&E)-stained lung sections taken from Birc3�/� mice revealed

signs of necrotizing bronchitis (Figure 2), we turned our attention

to the effect cIAP2 deletion has on cell death following PR/8

nc.

Figure 1. Nonhematopoietic Expression of cIAP2 Is Required for Survival after Pulmonary Challenge with Influenza A Virus

(A and B) (A) Survival and (B) body weight changes of PR/8-infected Birc3�/� (n = 20) and WT (n = 33) mice compared to mock-inoculated WT mice (n = 15)

(p < 0.0001; log rank). Data are pooled from two independent experiments; each line in (B) represents an individual mouse.

(C) Influenza M1 gene expression (mean ±SEM) by qPCR at 3 or 5 days p.i. Data are pooled from two independent experiments (n = 8–10 mice per time point).

(D) Viral titers were quantified on 3, 7, and 11 days p.i. by plaque assay (mean ±SD; n = 3–4 mice per genotype per time point).

(E) Survival rate of chimeric mice after infection with PR/8 virus. Log rank statistical significance is depicted in the table below the graph.

(F and G) BALF IFN-a (F) and CXCL10 (G) during infection. Data are pooled from two independent experiments (mean ±SEM; n = 8–10mice per genotype per time

point). See also Figure S1.

Cell Host & Microbe

cIAP2 Maintains Lung Homeostasis during Infection

infection. Real-time qPCR analysis of death ligands revealed that

the expression of Fasl, Tnfsf10, and Tnf in the lung was induced

by infection in both Birc3�/� andWTmice but that Birc3�/� mice

exhibited higher levels of these transcripts than WT mice at later

time points (Figure 3A). However, when we examined apoptotic

caspase activation by immunofluorescence and immunoblot

analysis in lungs of infected Birc3�/� mice, we found reduced

levels of processed caspase-3 (Figures 3B and 3C) and cas-

pase-8 (Figure 3C) compared to WT controls. This difference

was observed despite the presence of inflammation, as

Cell H

measured by NF-kB2 activation (Figure 3C). Correspondingly,

the proportion of apoptotic pulmonary epithelial cells

was reduced in Birc3�/� mice compared to WTmice, as demon-

strated by fluorescence-activated cell sorting (FACS)

quantification of active caspase-3 in dying CD326+CD45�

CD31�Ter119� airway epithelial cells (AECs) isolated from colla-

genase-digested lungs of PR/8 infected mice at day 7 p.i. (Fig-

ures 3D–3F). Analysis of tissue sections by immunofluorescence

showed that CD326+ AECs in the bronchiole lumen of Birc3�/�

mice were negative for active caspase-3 (Figure 3G). No

ost & Microbe 15, 23–35, January 15, 2014 ª2014 Elsevier Inc. 25

Figure 2. Respiratory Tissue Damage Following Influenza Infection in Birc3–/– Mice

(A and B) H&E sections through themain bronchiole of the left lobe showing pulmonary hemorrhage (star) and epithelial shedding (black arrows) and degeneration

(black arrow head). Scale bar indicates 25 mm. Insets correspond to the boxed regions. Images shown are representative of four to five mice per genotype for

three independent experiments.

(C) BSA-FITC arbitrary fluorescent units in BALF (mean ±SD; n = 4–5 mice per genotype per time point). One-way ANOVA with Bonferroni’s post-test (*p < 0.05).

(D and E) Epithelial cell marker CD326 (D) or PR/8 virus antigens (E) immunofluorescence. White arrowheads point to epithelial cell shedding. Images shown are

representative of two to three mice from two independent experiments. Scale bar indicates 50 mm.

Cell Host & Microbe

cIAP2 Maintains Lung Homeostasis during Infection

differences in CD45+ immune cell (Figure 3E, bottom) or

CD31+CD45� lung endothelial cell (Figure S2A, related to Fig-

ure 3) apoptosis could be detected in infected Birc3�/� mice

compared with WT mice, suggesting that modulation of cell

death in the absence of cIAP2 is cell-type specific.

The NLRP3-caspase-1 inflammasome has been reported to

protect the host from influenza-induced acute injury to the respi-

ratory epithelium (Thomas et al., 2009). On the other hand,

hyperactivation of the inflammasome pathway through loss of

Ripk2 was recently linked to influenza-induced airway neutro-

philia, necrotic cell death, tissue damage, and lethality (Lupfer

et al., 2013). Because cIAP2 is required for efficient activation

of caspase-1 (Labbe et al., 2011) and RIPK2 ubiquitination (Ber-

trand et al., 2009), we next asked what role, if any, activation of

caspase-1 and its ensuing pyroptotic cell death program might

play in the lethality to influenza in Birc3�/� mice. At the sublethal

dose of PR/8 used in this study, Casp1�/�/Casp11�/� and

Nlrp3�/� mice proved as resistant to infection as WT mice (Fig-

ure S2B, related to Figure 3). In contrast, deficiency in either

the inflammasome adaptor ASC or overall IL-1 signaling

26 Cell Host & Microbe 15, 23–35, January 15, 2014 ª2014 Elsevier I

(Il1r1�/�) impaired protection to PR/8 infection (Figure S2B).

However, the frequency of necrotic AECs in both of these geno-

types was either equivalent to or lower than WT mice (Fig-

ure S2C), suggesting that the pathogenic mechanisms at play

in Asc�/� and Il1r1�/� mice are distinct from those observed in

Birc3�/� mice and are acting independently of the canonical

inflammasome pathway in this model. Moreover, contrary to

Birc3�/� mice, infection of Ripk2�/� mice resulted in slightly

higher BALF IL-1b and IL-6 levels (Figures S2D and S2E) and

increased lung neutrophil infiltration (Figure S2F), consistent

with previous data (Lupfer et al., 2013). In addition, Ripk2�/�

mice exhibited normal AEC apoptosis (Figure S2G). These re-

sults argue that the Birc3�/� mice phenotype is independent of

a deregulation of the autophagy processes controlled by the

NOD2-RIPK2 axis that lead to aberrant caspase-1 activation

and lethality in that system. Altogether, these findings suggest

that cIAP2 likely confers protection to influenza independently

of its function in caspase-1 activation.

Caspase-11-induced pyroptotic cell death, while a defense

strategy against bacterial pathogens that engage type I

nc.

Cell Host & Microbe

cIAP2 Maintains Lung Homeostasis during Infection

interferons (Rathinam et al., 2012), can also contribute to lethal

inflammatory disease independently of caspase-1 (Broz et al.,

2012). Caspase-11 was induced by PR/8 infection in both WT

and Birc3�/� mice (Figure S2H). To assess to what extent

caspase-11 might influence PR/8-induced lethality, we ob-

tained, from two independent colonies, Birc3�/� mice that

were either carriers or not of a passenger intronic inactivating

deletion in the Casp11 gene (Kenneth et al., 2012) and inter-

crossed them to each other. The presence or absence of cas-

pase-11 did not alter the susceptibility of Birc3�/� mice, as the

mortality rates between Birc3�/�Casp11+/+, Birc3�/�Casp11+/�

and Birc3�/�Casp11�/� littermates were not different (Fig-

ure S2I). Additionally, caspase-11 deficiency did not render

mice more susceptible to intranasal infection with a sublethal

dose of a second influenza A strain (H3N2, Figure S2J), unlike

H3N2-infected Birc3�/� mice, which had reduced survival (Fig-

ure S2J) as well as increased AEC necrosis on day 7 p.i. (Figures

S2K and S2L). These results exclude a strain-inherent property

related to either increased infectivity and/or pathogenicity of

PR/8 in Birc3�/� mice and suggest that the activity of either

caspase-1 or caspase-11 is unlikely to account for the observed

lethality. Collectively, these findings demonstrate that a fraction

of the AEC cell death induced by influenza infection proceeds

independently of the activation of both apoptotic and inflamma-

tory caspases in Birc3�/� mice.

Necroptosis Inhibition inBirc3–/– Mice Improves SurvivalTo test whether necroptosis influenced the progression of influ-

enza-induced lethality, we used the chemical inhibitor Necrosta-

tin (Nec)-1 to block the kinase activity of RIPK1 in vivo, because

RIPK1-deficient mice exhibit early postnatal lethality (Kelliher

et al., 1998). Nec-1 prolonged the survival of Birc3�/� mice (Fig-

ure 4A; p = 0.0016) and diminished epithelial cell necrosis and

bronchial degeneration within the airways (Figure 4C), in marked

contrast with the tissue damage observed in vehicle-control-

treated mice (Figure 4B). Concordantly, genetic deletion of

Ripk3 protected Birc3�/� mice from PR/8-induced lethality to

levels similar to those of Ripk3�/� and WT mice (Figure 4D),

and it restored airway epithelial architecture without impacting

hemorrhage (Figure 4E). This rescue was dependent on Ripk3

deletion from the radio-resistant compartment of Birc3�/�

mice, since reconstitution of Birc3�/�/Ripk3�/� mice with

Ripk3-sufficient hematopoietic cells did not modify resistance

to PR/8 (Table 1). Viral clearance was not affected inmice lacking

Ripk3, as demonstrated by quantification of PR/8 titers in lung

tissues (Figures 4F and 4G). These experiments provided

genetic evidence that necroptosis is not required for effective

antiviral host defense against influenza but, rather, is a patholog-

ical feature that drives elevated morbidity and mortality in

infected Birc3�/� mice. To elucidate the potential mechanisms

by which cIAP2 deficiency skews the cell death programs in

AECs toward necroptosis following PR/8 infection, we immuno-

blotted lung homogenates for all known components that enable

RIPK3-dependent necrosis. The short isoform of FLIP (FLIPS)

inhibits apoptosis while actively promoting RIPK3-dependent

necrosis, whereas FLIP long (FLIPL) antagonizes both types of

cell death (Green et al., 2011). Because of higher FLIPS expres-

sion and a decreased FLIPL:FLIPS ratio in lungs of viral-infected

Birc3�/� mice (Figure 4H), we hypothesized that lack of cIAP2

Cell H

might promote the formation of the necrosome complex consist-

ing of RIPK1, RIPK3, and FADDduring influenza infection. To test

this, we immunoprecipitated endogenous FADD from lung ex-

tracts and examined the assembly of the necrosome. Although

RIPK3 was constitutively associated with FADD, as previously

reported (Cho et al., 2009), influenza infection led to additional

recruitment of RIPK3 to this FADD complex in Birc3�/� mice

compared to WT infected mice (Figure 4I, lanes 10–14 and

5–9; p = 0.0317), whereas virus-dependent interaction of

RIPK1 with FADD was remarkably similar between genotypes

(Figure 4I). In naivemice, a RIPK1-FADD complex spontaneously

assembled upon cIAP2 depletion (Figure 4I, lanes 3 and 4 and 1

and 2), as noted by others (Feoktistova et al., 2011; Tenev et al.,

2011). Our data suggest that cIAP2 deficiency favors the as-

sembly of the necrosome during infection, which translates to

a skewing of cell death toward necroptosis. Pharmacological

depletion of cIAPs in C57BL/6 by treatment with a SMAC

mimetic also resulted in reduced survival to PR/8 infection (Fig-

ures S3A and S3B, related to Figure 4) and a notable increase in

AEC necrosis on day 7 p.i. (Figures S3C and S3D). Altogether,

these data indicate that, following influenza virus infection,

cIAP2 expression in the lung determines, in a cell-specific

fashion, the sensitivity to necroptosis.

Role of Death Ligands in the Development ofNecroptosis-Induced Lethality in Birc3–/– MiceOne function of cIAPs is to act as negative regulators of caspase-

independent Fas-induced cell death (Geserick et al., 2009). We

therefore reasoned that cIAP2 deficiency might affect lung

homeostasis following infection by disturbing DR-mediated

signaling in AECs, rather than through direct virus-induced

sensing or cytopathic effects. Because of the observation that

influenza-induced acute lung injury stems from the absence of

cIAP2 expression in the nonhematopoietic compartment (Fig-

ure 1E and Table 1), and because abrogation of death ligand

expression by immune cells attenuates lung epithelial apoptosis

following influenza virus pneumonia (Herold et al., 2008), we

explored the role of DR signals through bone marrow transplan-

tation experiments. To this end, marrow from Faslgld, Tnfsf10�/�,Tnftm1Gkl, and Ltatm1Dch were transferred into irradiated Birc3�/�

mice to block specific DR signaling (Table 1). Micewith disrupted

Tnf (Figure 5A) or lymphotoxin-a (Figure 5B) signaling developed

influenza-induced lethality with identical kinetics and severity

compared to Birc3�/� mice transplanted with WT marrow

(WT/Birc3�/�), whereas genetic mutation of FasL (Figure 5C)

from the immune compartment had a stronger effect than TRAIL

deficiency (Figure 5D and Table 1) in protecting Birc3�/� mice

following PR/8 infection. To address whether direct infection of

AECs was required for necrosis induction in vivo, we analyzed

by FACS the proportion of uninfected AECs (measured as nega-

tive for PR/8 nucleoprotein; Figure 5E) that on day 7 p.i. stained

positive for viability dye uptake without caspase-3 activation

and/or phosphatidylserine translocation. Necrosis was induced

in both uninfected and infected AECs from PR/8-challenged

mice; however, the frequency of necrotic cells in Birc3�/� mice

exceeded that in WT mice (Figure 5F), regardless of whether

the cells were infected or not, revealing that skewing of AEC

cell death in the absence of cIAP2 is linked to airway inflamma-

tion rather than direct virus infection of AECs. These data

ost & Microbe 15, 23–35, January 15, 2014 ª2014 Elsevier Inc. 27

(legend on next page)

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cIAP2 Maintains Lung Homeostasis during Infection

28 Cell Host & Microbe 15, 23–35, January 15, 2014 ª2014 Elsevier Inc.

Cell Host & Microbe

cIAP2 Maintains Lung Homeostasis during Infection

suggest that FasL and (to a lesser extent) TRAIL signaling, as

opposed to direct viral cytopathic effects, are major determi-

nants of mortality triggered by cIAP2 deficiency during influenza

infection.

DISCUSSION

A cell’s decision to self-destruct in response to a virus is irrevers-

ible, and, therefore, in order to be an effective antiviral defense

strategy, it must be tightly controlled to maintain tissue homeo-

stasis during infectious disease. Similar to cell-fate decisions

regulated by XIAP in the liver (Jost et al., 2009), this work iden-

tifies a biological role for cIAP2 as a gatekeeper of pathological

DR-induced activation of cell death during respiratory infection.

We show that cIAP2 regulates death signaling following influenza

infection at the level of the assembly of the necrosome and the

stoichiometry of FLIP isoforms in the lung. RIPK3-dependent

programmed necrosis is the favored cell death mechanism

when the cellular apoptosis machinery is inhibited by FLIPS

(Feoktistova et al., 2011). Other IAPs appear to play nonredun-

dant roles in the infected lung as they were unable to compen-

sate for cIAP2-deficiency, which increased susceptibility to

DR-induced necroptosis independently of pathogen burden or

inflammatory response.

In line with previous in vitro work (Feoktistova et al., 2011;

Tenev et al., 2011), spontaneous assembly of the RIPK1-FADD

ripoptosome under steady-state conditions is insufficient for

cell death induction in vivo. Conceivably, its formation in the

absence of cIAP2 in the lung only preconditions AECs to become

more sensitive to death ligands, with the death-inducing sensi-

tivity of this complex once formed being modulated by the

expression levels of FLIP isoforms, as reported by others (Feok-

tistova et al., 2011; Oberst et al., 2011). Whereas necroptosis

acts as an antiviral mechanism against some DNA viruses (Cho

et al., 2009; Upton et al., 2010), it is dispensable for the control

of influenza replication, arguing for case-specific use of this

death modality during immune defenses to viruses. It is aston-

ishing that a minor switch in the decision of the type of death

by which AECs self-destruct can account for the extensive

epithelium loss that is characteristic ofBirc3�/� lungs. But a rela-

tively small increase of �10% in AEC death is sufficient to result

in considerable cell loss over time and leads to severe virus-

induced acute lung injury (Sanders et al., 2013). Necrotic loss

of alveolar type II epithelial cells, which are sensitive to FasL (Ha-

mann et al., 1998) and have an important role in the endogenous

repair capacity of the alveolar epithelium (Wang et al., 2010),

Figure 3. Caspase-Independent Cell Death of Birc3–/– AECs during Infl

(A) Tnfa, Tnfsf10, and Fasl expression in infected lungs (*p < 0.05, **p < 0.01; Stud

n = 8–10 mice per genotype per time point).

(B) Active caspase-3-stained lung sections from mock- or PR/8-infected mice (s

genotype for two independent experiments.

(C) Immunoblot analysis of lung tissue at 7 days p.i. (representative of two indep

(D and E) Representative FACS plots of day 7 airway epithelium dead cell number

Live/Dead stain (ViD) and (E) active caspase-3 staining; n = 8–10 mice total per g

(F) FACS quantification of caspase-dependent AEC cell death (mean ±SEM; Man

mice total for each genotype).

(G) CD326-positive cells in the bronchiole lumen of Birc3�/� mice are negative

representative of two to three mice per genotype for two independent experime

Cell H

could compromise repopulation of the injured epithelial lining

and accelerate AEC depletion. Our data also caution that deple-

tion of the cIAPs by synthetic IAP antagonists could adversely

affect individuals who develop respiratory viral infections. Taken

together, these observations favor a model in which cell death

initiation integrates the different stresses that converge on

lowering a cIAP expression threshold that otherwise effectively

prevents pathological RIPK3-dependent necrosis—specifically

during viral infection.

Sensitization of mucosal epithelial cells to TNF-induced nec-

roptotic death plays an important role for the development of

different pathologies in mucosal tissues (Gunther et al., 2011).

Surprisingly, although TNF was upregulated in Birc3�/� mice,

development of lethality following influenza infection was

rescued in mice lacking functional FasL or TRAIL signaling rather

than through Tnf/Lta. The potential importance of this pathway in

humans is hinted at by earlier human studies showing that

increased expression of Fas on the alveolar epitheliumwas asso-

ciated with death in cases of acute lung injury (Albertine et al.,

2002). Although we cannot fully exclude that endothelial cell

apoptosis, which was increased in Birc3�/� mice, could also be

involved in lesion development and pathogenesis as previously

reported in models of pulmonary hemorrhagic shock (London

et al., 2010; Lu et al., 2004), the rescue of lethality (but not of hem-

orrhaging) through pharmacological inhibition of RIPK1’s kinase

activity or through genetic ablation of Ripk3 suggests that aber-

rant necroptosis accounts for the greater susceptibility of

Birc3�/� mice to influenza infection. These observations point

to the existence of context-sensitive and cell-specific death-

inducing ligands and regulatory mechanisms of RIPK3-depen-

dent necroptosis. Distinct from its role in necroptosis, RIPK1

kinase activity stimulates Sp1-dependent transcription of

TNF-a (Christofferson et al., 2012) and promotes IL-1a secretion

in response to deregulated SHP-1 activity in macrophages

(Lukens et al., 2013). Given that deficiency in TNF-a (Damjanovic

et al., 2011) or IL-1R1 signaling (Figure S2) sensitizes the host to

influenza infection, these observations also provide plausible

mechanistic insights as to why genetic codeletion of RIPK3 res-

cuesBirc3�/�mice, whereas they are only partly rescued by inhi-

bition of RIPK1. However, differentmechanismsmay be at play in

the partial protection offered by Nec-1: aside from its poor phar-

macokinetic characteristics in vivo and its off-target activity

(Degterev et al., 2005), RIPK1 kinase activity also participates in

apoptosis when cIAPs are depleted (Wang et al., 2008), and,

in certain conditions, RIPK3-dependent necroptosis can

occur in the absence of RIPK1 (Vanlangenakker et al., 2011a).

uenza Infection

ent’s t test). Data are pooled from two independent experiments (mean ±SEM.;

cale bar indicates 200 mm). Images are representative of two to three mice per

endent experiments). Each lane corresponds to an individual mouse.

s (D), as gated on CD326+CD45�CD31�Ter119� and defined as positive for the

enotype.

n-Whitney test). Data are pooled from two independent experiments (n = 8–9

for active caspase-3 (white arrows; scale bar indicates 200 mm). Images are

nts. See also Figure S2.

ost & Microbe 15, 23–35, January 15, 2014 ª2014 Elsevier Inc. 29

(legend on next page)

Cell Host & Microbe

cIAP2 Maintains Lung Homeostasis during Infection

30 Cell Host & Microbe 15, 23–35, January 15, 2014 ª2014 Elsevier Inc.

Table 1. Influenza-Induced Lethality in Bone-Marrow Chimeric cIAP2-Deficient Mice after Intranasal Infection with PR/8 Virus

Survived Died Total Survival

Disadvantage

Survival

Benefit

p Valuea Versus

WT>WT

p Valuea Versus

WT>Birc3�/�Number % Number % Number

WT>WT 48 72.7 18 27.3 66 � �WT>Birc3�/� 3 9.1 30 90.9 33 + � <0.0001

WT>Birc3�/�Ripk3�/� 5 54.5 6 45.5 11 � + NS 0.0012

WT>Ripk3�/� 11 68.8 5 31.3 16 � + NS <0.0001

Faslgld > Birc3�/� 10 62.5 6 37.5 16 � + NS <0.0001

Tnfsf10�/�>Birc3�/� 4 44.4 5 55.6 9 � + NS 0.0116

Tnf�/�>Birc3�/� 2 20.0 8 80.0 10 + � 0.0011 NS

Lta�/�>Birc3�/� 0 0 6 100 6 + � 0.0003 NSaChi-square test comparing survival distribution at day 14 p.i. with 0.4LD50 of PR/8.

Cell Host & Microbe

cIAP2 Maintains Lung Homeostasis during Infection

The cIAPs have previously been implicated in antiviral immune

signaling. While RNAi depletion of cIAP2 impaired RIG-I-

mediated sensing of Sendai virus or cytoplasmic poly(I:C) (Mao

et al., 2010), we found no intrinsic defect in RIG-I signaling in

primary Birc3�/� BMDCs. In fact, we found normal innate and

CD8+ T cell antiviral responses to influenza virus in Birc3�/�

mice. Moreover, in spite of reduced neutralizing antibody

responses, Birc3�/� mice had similar lung virus titers to WT con-

trols, establishing that virus replication does not correlate with

the lethal outcome of primary infection and that cIAP2 does

not play an essential part in controlling it. Despite reports of

increased mortality in influenza-infected inflammasome-

deficient mice (Allen et al., 2009; Ichinohe et al., 2009; Thomas

et al., 2009), lethality inCasp1�/�/Casp11�/� orNlrp3�/� animals

was similar to that ofWT controls, as recently observed by others

(Jamieson et al., 2013). In addition, inflammasome-deficient

mice had either normal or lower necrotic AEC death levels, in

contrast to what was observed for Birc3�/� mice. The accumu-

lated evidence reinforces that, in the context of influenza infec-

tion, cIAP2 promotes host resistance independently of its known

roles in caspase-1 activation, immune cell signaling, or

development.

Overall, influenza-induced lethality in Birc3�/� mice manifests

primarily as an impairment in DR-induced cell death control that

does not hinder immune-mediated virus control but rather unbal-

ances death ligand signaling output in favor of necrosis of

infected but also of bystander uninfected cells. This is consistent

Figure 4. Inhibiting RIPK3-Dependent Programmed Necrosis Prevents

(A) Survival rate of infected Birc3�/� mice treated with Nec-1 or vehicle intrape

experiments.

(B and C) H&E-stained lung cross-sections of Birc3�/� vehicle-treated (B) and N

within the bronchioles (arrow head: erosion). Insets correspond to the boxed re

mouse from two independent experiments with n = 3–4 mice per group.

(D) Survival rate of Birc3�/�, Birc3�/�Ripk3�/�, Ripk3�/� and WT mice to PR/8 in

(E) H&E-stained lung cross-sections on 11 days p.i. Data are representative of tw

scale.

(F) Viral loads by plaque assay (mean ±SD; n = 4 mice per genotype per time po

(G) Lung sections stained for PR/8 antigens (scale bar indicates 50 mm).

(H) Immunoblot analysis of necrosome component expression in lung tissue from

individual mouse. Densitometry analysis using ImageJ software (mean ±SEM; n =

infected mice per genotype; *p = 0.0315, Mann-Whitney test).

(I) Formation of the necrosome on 7 days p.i. Asterix denotes (IgGH) heavy chains.

FADD ratio is shown at the bottom. See also Figure S3.

Cell H

with the concept that insufficient host defense to infectious dis-

ease can be due to an impaired ability to tolerate tissue damage

(Medzhitov et al., 2012). In this context, we envisage that the

transcriptional induction of cIAP2 by inflammatory triggers

(Wang et al., 1998) may have evolved to ensure survival of critical

cell types in response to strong DR signals induced by inflamma-

tion. The fact that the lethality from viral-induced necrosis in the

context of cIAP2 deficiency is found for two different influenza

strains points to a more general mechanism that is not specific

to a single virus and, thus, is likely of broader relevance for our

understanding of the mechanisms that are engaged during

airway inflammation to maintain tissue homeostasis and host

fitness. Indeed, DR-mediated lung epithelial injury is not

restricted to influenza virus, as it also occurs during infection

with other respiratory RNA viruses (Bem et al., 2010). Therefore,

continued characterization of cytoprotective tolerance mecha-

nisms, like the one described here during influenza infection,

may provide novel entry points for therapeutics against acute

lung injury.

EXPERIMENTAL PROCEDURES

Reagents

ELISA kits for anti-CXCL10 (DY466) were from R&D and VeriKine anti-IFNa

(42120) were from PBL; anti-caspase-8 Asp387 (9429) and anti-cFLIP (3210)

from Cell Signaling Technology; anti-FADD (M-19) was from Santa Cruz;

anti-RIPK1 (610459) was from BD Transduction Laboratories; anti-RIPK3

(PSC-2283) was from Axxora; anti-active caspase-3 (AF835) was from R&D;

Influenza-Induced Lethality in Birc3–/– Mice

ritoneally daily starting on 3 days p.i. Data are pooled from two independent

ec-1-treated (C) mice on 7 days p.i. Black arrow indicates epithelial shedding

gions (enlarged by an 8.53 zoom). Each section corresponds to an individual

fection. Data are pooled from two independent experiments.

o independent experiments with n = 3–4 mice per group. All H&E are to 25 mm

int).

PR/8-infected mice (lysates used in immunoprecipitation in (I). Each lane is an

3 independent experiments with each two to three uninfected and four to five

Each lane is an individual mouse. Densitometry analysis of RIPK1 and RIPK3 to

ost & Microbe 15, 23–35, January 15, 2014 ª2014 Elsevier Inc. 31

Figure 5. FasL and TRAIL Are Required for Influenza-Induced

Lethality in Birc3–/– Mice

(A–D) Survival rate of chimeric mice after PR/8 infection. Data are pooled from

two independent experiments in (D).

(E) Representative FACS plots of infected (NP+) versus noninfected

(NP�) AECs.(F) FACS quantification of cell-death type (necrosis, open circles; apoptosis,

closed circles) after 7 days p.i. in infected (NP+) versus uninfected (NP�) AECsfrom the same mouse. Necrosis (open circles) is defined as annexin V�, activecaspase-3� and ViD+; apoptosis (closed circles) is defined as annexin V+,

active caspase-3+, and ViD+/�. Lines and bars represent the mean and SD

(n = 5–6 mice per genotype, *p < 0.05; **p < 0.001; Student’s two-tailed t test).

Cell Host & Microbe

cIAP2 Maintains Lung Homeostasis during Infection

32 Cell Host & Microbe 15, 23–35, January 15, 2014 ª2014 Elsevier I

anti-influenza A,B (M149) was from Takara Bio; anti-CD326 (G8.8), anti-active

caspase-3-PE (C92-605), and streptavidin APCCy7 were from BD Biosci-

ences; Alexa Fluor-594 or Alexa Fluor647 anti-rabbit or rat antibodies were

from Molecular Probes/Life Technologies; anti-CD31-APC (390), anti-CD45-

PECy7 (30-F11), anti-CD326-PerCP-eFluor710 (G8.8), and anti-Ter119 were

from eBioscience; anti-influenza A virus nucleoprotein-FITC (431) was from

Abcam; PR/8 was from Charles River; Nec-1 (N9037), BSA-FITC (A9771),

and protein G sepharose beads (P3296) were from Sigma-Aldrich; Smac-

mimetic LCL-161 (A-1147)was from Active Biochem; and SURFLO I.V.

20G31’ catheter (SR-0X2025CA) was from Terumo (Elkton).

Mice

All mice were maintained at McGill University and were previously described

(Conte et al., 2006; Glaccum et al., 1997; Kobayashi et al., 2002; Kuida

et al., 1995; Mariathasan et al., 2004, 2006; Newton et al., 2004).

Birc3�/�Ripk3�/� mice were generated through intercrossing. B6Smn.C3-

Faslgld/J (001021), B6.129S-Tnftm1Gkl/J (005540), B6.129S2-Ltatm1Dch/J

(002258), and B6.SJL-PtprcaPep3b/BoyJ (CD45.1 congenics, 002014) were

from The Jackson Laboratory. Tnfsf10�/� mice from Amgen (Seattle) (Sedger

et al., 2002) were maintained at the University of Minnesota. Animals were

used at 6–8 weeks old or 12–13 weeks old for the chimeras. All animal exper-

iments were approved by the McGill University Animal Care and Ethics

Committee in accordance with the guidelines of the Canadian Council on

Animal Care.

Chimeras

Mice lethally irradiated with a dose of 1,000 rads on a X-Ray RS-2000 Biolog-

ical irradiator were injected intravenously with red blood cell-depleted bone

marrow from the indicated sex-matched donors. The extent of engraftment

was verified after 6 weeks by FACS staining of blood lymphocytes with

anti-CD45 and anti-B220 and by genotyping. In control experiments

using congenic CD45.1 and CD45.2 mice, chimerism was usually found to

be R98% as assessed in the lung and blood.

Virus Infection and Lung Tissue Sampling

Age- and sex-matched mice were anesthetized intramuscularly with xylazine

and ketamine and infected intranasally with a sublethal (0.4LD50; 12.9 HAU/

mouse) weight-adjusted dose of PR/8 in 15 ml of PBS. Where indicated,

Nec-1 was administered daily intraperitoneally at 5 mg kg�1 on days 3–14

p.i. LCL-161 was administered by oral gavage twice weekly at 50 mg kg�1

or otherwise stated in figure legends. After euthanasia by CO2, the trachea

was exposed, cannulated, and BAL was collected by injecting and pooling

two volumes of 0.5 ml cold, sterile PBS. The left lung lobes were used for

histopathological analysis. The right lungs were used for RNA isolation and/

or homogenized and sonicated in B150 buffer (20 mM Tris-HCl [pH 8.0],

150 mM KCl, 10% glycerol, 5 mM MgCl2, and 0.1% NP-40 supplemented

with a protease inhibitor cocktail [11836153001; Roche] and phosphatase

inhibitors), followed by boiling in Laemmli sample buffer for immunoblot. For

epithelial integrity measurements, BSA-FITC was administered intravenously

at 1 mg kg�1 2 hr before killing, following which fluorescence content in

BALF was measured on a FluoStar OPTIMA at 420 nm absorbance and

520 nm emission wavelengths.

nc.

Cell Host & Microbe

cIAP2 Maintains Lung Homeostasis during Infection

Histopathology and Immunofluorescence

Lung lobes were collected in 10% neutral-buffered formalin, and tissue

sections were prepared from paraffin block and stained with H&E. Immuno-

fluorescence slides were analyzed on a Zeiss Axioskop upright wide-field

microscope (2030.5 or 403/0.75 Plan-Neofluar objectives) equipped with a

high-resolution monochromatic AxioCam HRm camera and driven by

AxioVision version 4.9.1 (Carl Zeiss Microscopy).

qPCR

Total lung RNA was extracted using Trizol reagent, and reverse transcription

was performed on 1 mg total RNA in a final volume of 20 ml using random

hexamers and the M-MLV RT kit (all from Invitrogen). qPCR reactions were

performed using 2 ml of cDNA, 12.5 ml of the iTaq SYBR green supermix

(Biorad), and 5 mM of each primer (Table S1) in a total volume of 25 ml. An

ABI PRISM 7900 Sequence Detection System (Applied Biosystems)

programmed with an initial step of 3 min at 95�C, followed by 45 cycles of

15 s at 95�C, 30 s at 57�C and 33 s at 72�C. All reactions were run in duplicate,

averaged, and normalized to the housekeeping gene L32 to quantify the

relative gene expression from that of the mock-infected control using the

2�DDCT method.

Virus Titration by Plaque-Forming Unit Assay

Serial dilutions of lung homogenates were performed in PBS and used to infect

confluent MDCK cells for 1 hr at 37�C and 5% CO2. Following this period, the

mediumwas replacedwith 0.65%agarose in DMEMcontaining 0.2%BSA and

1 mg/ml TPCK-treated trypsin (Worthington). Infection was pursued for

3–4 days, and plaques were visualized by crystal violet staining.

Immunoprecipitation

For endogenous coimmunoprecipitation studies, whole-lung lysates (1 mg

protein) in B150 were cleared by incubation with protein G beads, incubated

with 1.5 mg anti-FADD antibodies for 2.5 hr at 4�C, and protein G beads

were added for an additional hour. Immunoprecipitates were eluted by boiling

in Laemmli buffer, electrophoresed on 10% SDS polyacrylamide gels, and

processed for immunoblot analysis using antibodies to mouse RIPK1,

RIPK3, and FADD.

FACS Assays

Collagenase-digested lung cells were surface stained with the appropriate

fluorochrome-labeled antibodies and the amine reactive viability dye LIVE/

DEAD aqua fixable stain (ViD, Molecular Probes) for 20 min in the dark at

4�C. Annexin V-eFluor450 (eBioscience) was added for 15 min at room

temperature after cell-surface FACS staining, followed by intracellular staining

for active caspase-3 and anti-influenza nucleoprotein, after which cells were

fixed in PBS containing 1% formaldehyde and stored at 4�C in the dark until

FACS analysis. Data were acquired on a Canto instrument (BD Biosciences)

equipped for the detection of eight fluorescent parameters. Data analysis

was performed using FACS DiVa version 6.0 software.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism software v5.0 and

SigmaPlot 12 statistical package. Data are represented as mean ± SD or

SEM. Comparisons between groups were performed with a two-tailed

Student’s t test or Mann-Whitney U test, one-way ANOVA test, or chi-square

test. Survival curves were analyzed with a log rank test.

SUPPLEMENTAL INFORMATION

Supplemental Information includes three figures, one table, and Supplemental

Experimental Procedures and can be found with this article online at http://dx.

doi.org/10.1016/j.chom.2013.12.003.

ACKNOWLEDGMENTS

We thank V. Dixit and Genentech for providing Ripk3�/�, Nlrp3�/�, Asc�/�,Casp11�/� mice; R. Korneluk for Birc3�/� mice and caspase-11 PCR geno-

typing protocol; P. Barker for Birc3�/�Casp11+/+ mice; R. Flavell for Ripk2�/�

and Casp1�/� mice; M. Divangahi for the NP366–374 tetramer; D. Lamarre for

Cell H

purified PR/8 virus; and J. Rinz for animal husbandry. This work was supported

by grants from the Canadian Institutes for Health Research (CIHR-MOP 79410)

and the Burroughs Wellcome Fund to M.S., who is a Fonds de Recherche en

Sante du Quebec (FRSQ) Senior Investigator and a McGill University William

Dawson Scholar. I.G.R.G. is supported by postdoctoral fellowships from the

FRSQ, CIHR, and by the Strauss Foundation. K.L., J.D., and A.S. are sup-

ported by CIHR studentships/fellowships. M.D. is supported by a FRSQ

studentship, and A.M. is supported by a CIHR/CAG/Abbott fellowship.

E.L.B. is supported by a postdoctoral fellowship from the Kidney Cancer

Association and National Institutes of Health grant CA109446 (to T.S.G.).

Received: June 17, 2013

Revised: November 10, 2013

Accepted: December 10, 2013

Published: January 8, 2014

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