TLR3, TRIF, and Caspase 8 Determine
Double-Stranded RNA-Induced Epithelial
Cell Death and Survival In Vivo
This information is current as
of June 18, 2017.
Christopher S. McAllister, Omar Lakhdari, Guillaume
Pineton de Chambrun, Mélanie G. Gareau, Alexis Broquet,
Gin Hyug Lee, Steven Shenouda, Lars Eckmann and Martin
F. Kagnoff
Supplementary
Material
References
Subscription
Permissions
Email Alerts
http://www.jimmunol.org/content/suppl/2012/12/03/jimmunol.120275
6.DC1
This article cites 67 articles, 34 of which you can access for free at:
http://www.jimmunol.org/content/190/1/418.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2012 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
J Immunol 2013; 190:418-427; Prepublished online 3
December 2012;
doi: 10.4049/jimmunol.1202756
http://www.jimmunol.org/content/190/1/418
The Journal of Immunology
TLR3, TRIF, and Caspase 8 Determine Double-Stranded
RNA-Induced Epithelial Cell Death and Survival In Vivo
Christopher S. McAllister, Omar Lakhdari, Guillaume Pineton de Chambrun,
Mélanie G. Gareau, Alexis Broquet,1 Gin Hyug Lee,2 Steven Shenouda, Lars Eckmann, and
Martin F. Kagnoff
T
he mucosa of the small intestine contains finger-like projections termed villi that are lined by a single-cell layer of
intestinal epithelial cells (IECs) that separate the intestinal lumen from the underlying immune cell-rich lamina propria.
IECs are armed with numerous pattern recognition receptors
(PRRs) that detect and activate host responses to protect against
viral and bacterial pathogens and their products (1). dsRNA is a virusassociated molecular pattern that is recognized by the endosomal
PRR TLR3, and intracellular cytoplasmic retinoic acid-inducible
gene (RIG)–like sensors (2, 3). Interestingly, dsRNA signaling
through TLR3 has been reported to induce small intestinal mucosal
damage in mice (4).
Department of Medicine, University of California, San Diego, La Jolla, CA 92093
1
Current address: Medicine Faculty, University of Nantes, Nantes, France.
2
Current address: Department of Internal Medicine, University of Ulsan College of
Medicine, Asan Medical Center, Songpa-gu, Seoul, Korea.
Received for publication October 2, 2012. Accepted for publication November 5,
2012.
This work was supported by National Institutes of Health Grants DK35108 and
DK80506 and by a grant from the William K. Warren Foundation.
Address correspondence and reprint requests to Prof. Martin F. Kagnoff, Department
of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA
92093-0623. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: BAX, Bcl-2–associated X; DC, dendritic cell;
DTR, diphtheria toxin receptor controlled by CD11c promoter; IEC, intestinal epithelial cell; IEL, intraepithelial lymphocyte; IKK, IkB kinase; IRF, IFN regulatory
factor; Isc, short-circuit current; MAVS, mitochondrial antiviral-signaling protein;
pIC, polyinosinic:polycytidylic acid; PKR, double-stranded RNA-activated protein
kinase; PRR, pattern recognition receptor; RIG, retinoic acid-inducible gene; WT,
wild-type.
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202756
TLR3 signals through the adaptor molecule TRIF, whereas the
RIG-like RNA sensors, RIG-I and Mda5, signal through the adaptor
molecule mitochondrial antiviral signaling (MAVS, also known as
IPS-1) (2). These pathways activate IFN regulatory factor 3 (IRF3)
and NF-kB transcription factors and result in the production of
type I IFNs and proinflammatory cytokines. Furthermore, activation of host signaling pathways can induce apoptosis, likely as a
means to kill infected host cells to shut down virus production (5).
Although NF-kB can have both pro- and antiapoptotic effects
within cells, IRF3 has been shown to interact with Bcl-2–associated X (BAX) protein to directly induce apoptosis after viral infection (6). In addition, in vitro studies have shown that TRIF,
unlike the TLR adaptor protein MyD88, interacts with caspase 8
through the receptor interacting protein 1 signaling molecule to
directly induce apoptosis (7–10).
PRR signaling pathways can also induce TNF that can act upon
other cells to either induce survival through NF-kB signaling or
death through apoptosis or necroptosis (11). Interestingly, TNF
injection induces IEC apoptosis in vivo in mice (12). This effect is
dependent on TNFR-1 and more pronounced in the proximal than
distal small intestine, possibly because of the expression pattern of
the TNFR-1 (13). Signaling through TNFR-1 leads to the recruitment of a signaling complex that activates caspase 8, which then
mediates apoptosis by activating effector caspases including
caspase 3 (11).
We investigated the mucosal signaling pathways activated by
dsRNA that cause small intestinal damage in vivo. We report that
dsRNA rapidly induces intestinal villus epithelial cell apoptosis
and cell loss in the small intestine that results in marked villus
shortening and significant diarrhea, with subsequent recovery.
dsRNA-induced IEC apoptosis in vivo was strictly dependent on
TLR3 and TRIF signaling through caspase 8 in IECs, and independent of other dsRNA signaling pathways, as well as TNF, IL-15,
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
TLR3 signaling is activated by dsRNA, a virus-associated molecular pattern. Injection of dsRNA into mice induced a rapid, dramatic, and reversible remodeling of the small intestinal mucosa with significant villus shortening. Villus shortening was preceded by
increased caspase 3 and 8 activation and apoptosis of intestinal epithelial cells (IECs) located in the mid to upper villus with ensuing
luminal fluid accumulation and diarrhea because of an increased secretory state. Mice lacking TLR3 or the adaptor molelcule TRIF
mice were completely protected from dsRNA-induced IEC apoptosis, villus shortening, and diarrhea. dsRNA-induced apoptosis was
independent of TNF signaling. Notably, NF-kB signaling through IkB kinase b protected crypt IECs but did not protect villus
IECs from dsRNA-induced or TNF-induced apoptosis. dsRNA did not induce early caspase 3 activation with subsequent villus
shortening in mice lacking caspase 8 in IECs but instead caused villus destruction with a loss of small intestinal surface epithelium
and death. Consistent with direct activation of the TLR3–TRIF–caspase 8 signaling pathway by dsRNA in IECs, dsRNA-induced
signaling of apoptosis was independent of non-TLR3 dsRNA signaling pathways, IL-15, TNF, IL-1, IL-6, IFN regulatory factor 3,
type I IFN receptor, adaptive immunity, as well as dendritic cells, NK cells, and other hematopoietic cells. We conclude that
dsRNA activation of the TLR3–TRIF–caspase 8 signaling pathway in IECs has a significant impact on the structure and function
of the small intestinal mucosa and suggest signaling through this pathway has a host protective role during infection with viral
pathogens. The Journal of Immunology, 2013, 190: 418–427.
The Journal of Immunology
dendritic cells (DCs), NK cells, and other hematopoietic cells.
Interestingly, this pathway proved to be host protective because, in
the absence of downstream activation of caspase 8, dsRNA caused
marked destruction of the small intestinal mucosa and death.
Materials and Methods
Reagents
Low m.w. polyinosinic:polycytidylic acid (pIC) was from InvivoGen (San
Diego, CA) and used in all studies unless indicated otherwise. pIC sodium
salt was from Sigma-Aldrich (St. Louis, MO). R848, ODN 1826, and LPS
from Escherichia coli O111:B4 were from InvivoGen. pIC was heated to
50˚C for 10 min and allowed to cool to room temperature to anneal.
Cleaved caspase 3 (Asp175) and cleaved caspase 8 (Asp387) (D5B2) Ab was
from Cell Signaling Technology (Danvers, MA). Recombinant mouse TNF
was purchased from PeproTech (Rocky Hill, NJ).
Mouse strains and treatment
Histology and immunohistochemistry
Proximal small intestine starting 2 cm distal to the pylorus and continuing
for the first third of the small intestine was processed as Swiss rolls (32).
Distal small intestine Swiss rolls were taken from the distal third of the
small intestine. Tissues were fixed in 10% formalin and embedded in paraffin, and 5-mm sections were stained with H&E. For cleaved caspase 3
staining, samples were deparaffinized, subjected to Ag retrieval, and
incubated with Ab overnight, after which HRP-conjugated secondary Ab
was added for 2 h followed by incubation with 3,39-diaminobenzidine
(Vector Laboratories) and counterstaining with hematoxylin. For NK1.1
and CD11c staining, small intestine Swiss rolls were frozen in OCT
(Tissue-Tek). Frozen sections were fixed in neutral buffered formalin for
10 min, washed, and blocked with 3% BSA in PBS. For NK1.1 staining,
sections were incubated overnight with FITC-conjugated NK1.1 (PK136)
(Abcam) or IgG negative control mAb, and then washed and visualized.
For DC staining, sections were incubated overnight with CD11c Ab (BD
Pharmingen) or Armenian hamster negative control (AbD Serotec) and
then washed and stained with Cy3 goat anti-Armenian hamster (Jackson
ImmunoResearch Laboratories).
Microscopy analysis
Villus lengths were measured in each Swiss roll, and the 10 longest villi
were averaged to give a final mean villus length. Cleaved caspase 3–stained
cells that were completely black were counted as positive cells, and 10
representative villi were counted and averaged to provide a value for each
mouse. Samples were examined in our laboratory under an Olympus BX41
microscope. Digital images were captured using the PictureFrame Application 2.3 (Optronics).
Ussing chamber studies
Segments of proximal small intestine were cut along the mesenteric border
and mounted in Ussing chambers (Physiological Instruments, San Diego,
CA), exposing 0.09 cm 2 of tissue area to 4 ml circulating oxygenated
Ringer’s buffer maintained at 37˚C. The buffer consisted (in millimolars)
of 140 Na+, 5.2 K+, 1.2 Ca2+, 0.8 Mg2+, 120 Cl2, 25 HCO32, 2.4 H2PO42,
and 0.4 HPO422. In addition, glucose (10 mM) was added to the serosal
buffer as a source of energy, osmotically balanced by mannitol (10 mM) in
the mucosal buffer. Agar–salt bridges were used to monitor the potential
difference across the tissue and to inject the required short-circuit current
(Isc) to maintain the potential difference at 0. This was registered by an
automated voltage clamp and continuously recorded by computer. Baseline
Isc values were obtained at equilibrium, 15 min after the tissues were
mounted and expressed as mA/cm2. Conductance (G) was also determined
at baseline as an indicator of ion flux and expressed as mS/cm2 . FITClabeled dextran (4 kDa; Sigma-Aldrich) was used as a probe to assess
macromolecular permeability and was added (2.2 mg/ml final concentration) to the luminal buffer once equilibrium was reached. Serosal samples
(240 ml) were taken at 30-min intervals for 2 h and replaced with fresh
buffer to maintain constant volume. Fluorescence was measured by end
point assay (Victor43; PerkinElmer), and the flux of FITC–dextran from
the mucosa to the serosa was calculated as the average value of two
consecutive stable flux periods (60–90 and 90–120 min) and expressed as
pmol/cm2/h (33).
Cytokine detection
Serum samples were collected 1h after treatment and stored at 280˚C.
Cytokine measurements used the mouse ultrasensitive TNF kit or proinflammatory 7-plex kit from Meso Scale Discovery (Gaithersburg, MD),
and cytokine levels were determined using a Meso Scale Discover Sector
Imager 6000.
RNA extraction and quantitative real-time PCR
Total cellular RNA was extracted using TRIzol (Life Technologies, Carlsbad,
CA), according to the manufacturer’s instructions. One microgram of total
RNA was used for iScript (Bio-Rad Laboratories, Hercules, CA) reverse
transcription, and the cDNA was used for quantitative real-time PCR using
SYBR green Master mix (Applied Biosystems, Foster City, CA) and
GAPDH forward primer 59-ATCAACGACCCCTTCATTGACC-39 and
GAPDH reverse primer 59-CCAGTAGACTCCACGAGATACTCAGC-39
or IFN-b forward primer 59-CTGAGCAGCTGAATGGAAAG-39 and IFN-b
reverse primer 59-CTTGAAGTCCGCCCTGTAGGT-39. Denaturation was
5 min at 95˚C, followed by 40 cycles of amplication at 95˚C for 30 s and 60˚C
for 30 s using an ABI StepOnePlus (Applied Biosystems). IFN-b induction
was calculated using the DDCt method, and values were normalized to
PBS-treated mice.
NK and DC depletion
To deplete NK cells, mice were injected with 200 mg purified anti-NK1.1
(PK136) mAb (eBioscience, San Diego, CA) or control IgG2 24 h before
injection of pIC. NK1.1+ cell depletion was confirmed by immunohistochemistry as described above. For DC depletion, DTR (26) mice were treated
with 4 ng/g diphtheria toxin i.p. (Sigma-Aldrich) 24 h prior to pIC injection. DC depletion was confirmed by immunohistochemistry as described
above.
Bone marrow chimeras
To generate bone marrow chimeric mice, WT or TLR3 recipients were
exposed to a single lethal dose of 9 Gy total body irradiation from a [137Cs]
source. Six hours later, these mice were injected i.v. with 2–5 3 106 bone
marrow cells from TLR32/2 or WT mice. Mice were rested for 8 wk before use.
Statistical Analysis
Data are expressed as mean 6 SD. Two groups were compared using
unpaired Student t test. ANOVA with Bonferonni posttest was used for
multiple group comparisons. Significant differences were reported where
p , 0.05.
Results
dsRNA induces IEC apoptosis in the small intestine
We used the synthetic dsRNA viral nucleic acid mimic pIC as a
probe of the host’s antiviral strategies in the small intestine. Administration of pIC i.p. resulted in villus shortening in the proximal small intestine, which was maximal between 3 and 6 h after
injection and recovered thereafter (Fig. 1A–C). The dramatic reduction in villus length correlated with decreased numbers of IECs
per villus (Supplemental Fig. 1A–C). The relatively rapid loss of
IECs and ensuing recovery suggested a dsRNA-activated pathway
that causes IEC loss, perhaps as a host response to rid the epi-
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
IEC-specific caspase-8 knockout mice (casp8DIEC) were generated in our
laboratory by breeding Casp8fl mice (14) with villin-Cre mice (15) as
described by Günther et al. (16). Wild-type (WT) C57BL/6J, TNF2/2,
TRIFlps2/lps2 (TRIF2/2) (17), BAX2/2 (18), RAG12/2 (19), IL-62/2 (20),
and IL-1R2/2 (21) mice from The Jackson Laboratory, IL-152/2 mice (22)
from Taconic, and IFNAR2/2 (23), double-stranded RNA-activated protein kinase (PKR)2/2 (24), TLR32/2 (25), CD11c-diphtheria toxin receptor (DTR) (26), IkK-bDIEC (27), IkK-aDIEC (28), TLR42/2 (29),
IRF32/2 mice (30), MAVS2/2 (31), and casp8DIEC mice were maintained in specific pathogen-free conditions at University of California,
San Diego. Mice were given a single i.p. injection of 30 mg/kg pIC, 10 mg/
kg R848, 5 mg/kg ODN 1826, or 20 mg/kg LPS in sterile PBS or saline
solution, using sterile PBS or saline injection alone as a control. In one
series of experiments, pIC was injected in the retro-orbital sinus of
anesthetized mice. Recombinant TNF was injected i.p. Mice were sacrificed
at 3–6 h after injection unless otherwise indicated. All animal studies were
approved by the University of California, San Diego Institutional Animal
Care and Use Committee.
419
420
TLR3–TRIF–CASPASE 8 PATHWAY SIGNALS EPITHELIAL APOPTOSIS
thelium of virus-infected cells while concurrently maintaining a
relatively intact IEC layer.
To determine whether apoptosis explained the decreased numbers of IECs, we evaluated cleaved caspase 3 staining of small
intestinal tissue sections (Fig. 1D–F) (34). IEC apoptosis preceded
villus shortening with a peak at 2 h after pIC i.p. (Fig. 1 G). Apoptotic IECs predominately localized in the mid to upper villus
region, with little apoptosis in the lower villus region and no increase in apoptosis in either the crypt region (i.e., the site of epithelial stem cells) or the subepithelial lamina propria (Fig. 1E).
Cleavage of caspase 8, an initiator caspase, was also observed at
2 h posttreatment (Supplemental Fig. 1D, 1E). Apoptosis of villus
IECs and villus shortening were less pronounced but still significant in the distal small intestine (Supplemental Fig. 1F–I), likely
reflecting shorter villi with decreased numbers of IECs in the distal
small intestine.
pIC-treated mice had significant fluid accumulation in the small
intestine and diarrhea that peaked at 4–6 h and returned to normal
thereafter (Fig. 1H–J). To address the cause of this increase in
intestinal fluid accumulation, Ussing chamber studies were done
to measure the small intestinal secretory state and mucosal permeability. Baseline Isc, indicative of active ion secretion, was
elevated in mice treated with pIC compared with PBS controls
(Fig. 1K). In contrast, conductance indicative of ion permeability
(Fig. 1L) and FITC–flux indicative of macromolecular perme-
ability (Fig. 1M) were not significantly altered by pIC treatment.
Taken together, these data suggest that increased luminal fluid
accumulation in the proximal small bowel resulted from an increased secretory state in these mice in the context of intact tight
junctions and mechanisms of endocytosis.
We next investigated whether the mode of pIC delivery determined IEC apoptosis, because injection by the i.p. route suggested
the possible involvement of i.p. macrophages or other cell subsets
specific to the peritoneal cavity in inducing villus shortening and
IEC apoptosis. However, there was no dependence on i.p. injection,
as either i.p. or retro-orbital sinus injection of pIC induced significant villus shortening in the proximal small intestine and IEC
apoptosis (Supplemental Fig. 1J–M, 1N, 1O). Moreover, Cy3labeled pIC was detected in the lamina propria of proximal small
intestinal villi after retro-orbital injection (Supplemental Fig.
1P, 1R). Administration of pIC by oral gavage did not induce villus
shortening or IEC apoptosis (Supplemental Fig. 1 M–O).
TLR3-TRIF signaling is required for IEC apoptosis
We found that TLR3 was required for dsRNA-induced intestinal
damage as reported before by others (4). Indeed, TLR32/2 mice
were completely resistant to dsRNA-induced IEC apoptosis and
villus shortening (Fig. 2A, 2B, 2D, 2E). Furthermore, dsRNA did
not induce IEC apoptosis or villus shortening in mice lacking
TRIF, the downstream adaptor molecule for TLR3 (Fig. 2C, 2F).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 1. pIC induces IEC apoptosis and small intestinal villus shortening. H&E-stained sections of
proximal WT mouse small intestine 3 h after PBS (A)
or pIC (B) 30 mg/g i.p. Scale bar, 200 mm. (C) Mean
villus length at indicated times after pIC. (D) Cleaved
caspase 3–stained sections of proximal small intestinal
mucosa 2 h after PBS or pIC (E, F). Scale bars, 100
mm (D, E) and 25 mm (F). (G) Cleaved caspase 3–
labeled cells per villus at specified times after pIC.
Representative images of the peritoneal cavity of PBS
(H) or pIC (I) 30 mg/g i.p. injected mice at 6 h. (J)
Fluid weight per length of the entire small intestine
at the indicated times after pIC. Values are mean 6
SD, n = 3–6 mice/time. **p , 0.01 versus PBS. S =
solid contents, no fluid. Ussing chamber measurements
of short circuit current (Isc) (K), conductance, G (L),
and macromolecular permeability (M) to 4 kDa FITC–
dextran in proximal small intestine pieces isolated
from mice treated with PBS or 30 mg/g i.p. pIC for 3 h.
Graphs show mean 6 SEM, n = 5 samples/treatment
group.
The Journal of Immunology
421
Villus length and the number of IECs with cleaved caspase 3
staining per villus were quantified and confirmed the essential
involvement of TLR3–TRIF signaling in pIC-activated villus shortening and apoptosis in the proximal small intestine (Fig 2G, 2H).
Neither TLR32/2 nor TRIF2/2 mice developed small intestinal fluid
accumulation or diarrhea. There was no difference in IEC proliferation between WT and TLR3 2/2 mice after pIC, indicating
that pIC did not damage the proliferative capacity of crypt cells
(Fig. 2I–L).
We assessed the requirement for other RNA sensors to determine
whether they had a contributory role in pIC-activated IEC apoptosis
in the small intestine. MAVS (IPS-1), the signaling adaptor for
RIG-like receptors, was not required for villus shortening or
caspase 3 cleavage (Fig. 2M, 2N). PKR is an IFN-inducible protein
kinase that can act as a general translation inhibitor and induce
apoptosis in response to activation by dsRNA (35). Mice deficient
in PKR had similar levels of villus shortening and numbers of
caspase 3–cleaved cells as WT mice (Fig. 2M, 2N), indicating that
PKR-dependent translation inhibition or signaling was not required for pIC-activated IEC apoptosis and villus shortening.
Apoptosis of small intestinal IECs induced by TLR ligands
High levels of TNF result in IEC apoptosis (12) and, consistent
with this, i.p. injection of 5 mg, but not 1 mg, of TNF induced IEC
apoptosis in WT mice (Fig. 3E–G). Because several TLR ligands
that signal through MyD88 induce the release of TNF (2), we
evaluated IEC apoptosis induced by other TLR ligands and found
that pIC induced significantly higher levels of IEC apoptosis than
the TLR7 ligand R848, TLR9 ligand ODN, and TLR4 ligand LPS
(Fig. 3 A–D, 3G). Moreover, pIC induced TNF levels orders of
magnitude lower than those TLR ligands, with LPS inducing the
highest TNF levels (Fig. 3H). Although others observed higher serum TNF levels after pIC (4) we established that this was likely due
to endotoxin contamination of pIC in their studies. However, we
found such levels of endotoxin contamination did not significantly
affect IEC apoptosis or villus shortening (Supplemental Fig. 2).
pIC-induced IEC apoptosis is independent of TNF, IL-6, and
IL-1 signaling
We definitively excluded the involvement of TNF in pIC-induced
apoptosis using mice deficient in TNF (TNF 2/2 ) that did not
produce TNF following dsRNA treatment (Fig. 3L). TNF2/2
mice were as sensitive as WT mice to dsRNA-induced IEC apoptosis (Fig. 3I–K). These results confirmed that TNF has no role in
pIC-induced small intestinal IEC apoptosis. We also confirmed
that IL-6 and IL-1b, two cytokines that were increased after pIC,
are not required for dsRNA-induced IEC apoptosis using IL-62/2
and IL-1R2/2 mice (Supplemental Fig. 3).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 2. TLR3–TRIF signaling is required for pIC-induced small intestinal villus shortening and IEC apoptosis. Cleaved caspase 3–stained sections
of proximal small intestine from PBS (A)–(C) and 30 mg/g pIC (D)–(F) i.p. treated WT, TLR32/2, and TRIF2/2 mice at 3 h. Scale bar, 100 mm. Villus
length (G) and cleaved caspase 3–labeled cells per villus (H) after PBS or 30 mg/g pIC i.p. at 3 h. Data are mean 6 SD, n = 3–4 mice/group. **p , 0.01
versus PBS-treated mice and pIC treated TLR32/2 and TRIF2/2 mice. BrdU-stained sections from the proximal small intestine 6 h after PBS i.p. (I) or 30
mg/g pIC i.p. (J) in WT mice, PBS i.p. (K), or 30 mg/g pIC i.p. (L) in TLR32/2 mice. Scale bar, 200 mm. (M) Villus length of MAVS+/+, MAVS2/2, WT, and
PKR2/2 mice at 3 h after 30 mg/g pIC i.p. (N) Cleaved caspase 3–labeled cells per villus for pIC treated MAVS+/+, MAVS2/2, WT and PKR2/2 mice at 3 h
after 30 mg/g pIC i.p. Mean 6 SD, n = 5–8 mice/group.
422
TLR3–TRIF–CASPASE 8 PATHWAY SIGNALS EPITHELIAL APOPTOSIS
IEC apoptosis is independent of IL-15 and NK cells
IL-15 signaling and NK cell mediated killing were reported to be
required for dsRNA-induced small intestinal villus shortening and
epithelial cell injury (4). To evaluate the role of IL-15 in our
model, we used mice deficient in IL-15 production. IL-152/2 mice
demonstrated the same level of sensitivity to pIC as WT mice
(Fig. 4A–D). To assess the role of NK1.1+ cells in dsRNA-induced
IEC apoptosis, those cells were depleted using Ab to NK1.1. We
observed the same level of IEC apoptosis and small intestinal
damage in NK1.1-depleted mice as in littermates receiving control
IgG (Fig. 4E–H). NK1.1+ cell depletion was confirmed by immunohistochemistry (Supplemental Fig. 4A–C).
IEC apoptosis does not require type I IFN signaling or IRF3
IFN-b expression in the small intestinal mucosa was increased in
WT but not TLR32/2 mice after pIC (Fig. 4I). Because type I IFN
signaling was reported to potentiate virus-induced apoptosis (36),
we assessed the involvement of type I IFN signaling in pIC-induced
IEC apoptosis and villus shortening. There was no significant difference between IFNAR2/2 and WT mice in villus shortening or
apoptosis (Fig. 4J–M).
Antiviral signaling through MAVS can activate IRF3 in
a manner that results in IRF3 interacting with BAX and localizing
to mitochondria to induce apoptosis (6). Because TLR3 signaling
also activates IRF3 (37), we tested whether this pathway was
involved in pIC-activated IEC apoptosis. We found no evidence
to suggest the involvement of IRF3 or BAX, because neither
IRF32/2 nor BAX2/2 mice manifested differences in villus length
or IEC apoptosis compared with WT mice after pIC treatment
(Fig. 4N–R).
NF-kB signaling in IECs does not affect villus IEC apoptosis
but is required to protect IECs in the small intestinal crypts
from apoptosis
TLR3–TRIF signaling can activate the transcription factor NF-kB
(37). To assess the involvement of NF-kB signaling in pICinduced apoptosis of IECs, we used mice conditionally deficient
in IkB kinase (IKK)a and IKKb in small intestinal IECs (27, 38).
The magnitude of IEC apoptosis in the villi of IKKa/bDIEC and
control mice was similar after pIC (Fig. 5B, 5E), whereas IKKa/
bDIEC mice had increased levels of IEC apoptosis in the small
intestinal crypts (Fig. 5B, 5F). To assess the contribution of IKKa
and IKKb to this phenotype, we used IKKaDIEC and IKKbDIEC
mice deficient selectively in IKKa or IKKb, respectively, in the
IECs and determined that increased crypt apoptosis after pIC was
dependent on the absence of IKKb signaling (Fig. 5D, 5F). Notably, this same pattern of IKKb dependence for crypt, but not
IEC, apoptosis was observed in response to TNF (Fig. 5G–J).
TLR3 signaling in cells of nonhematopoietic origin is required
for IEC apoptosis
NK1.1 cell depletion demonstrated that NK1.1+ cells were not
involved in pIC-activated IEC apoptosis (Fig. 4E–H). To exclude
the contribution of other immune cell subsets, we assessed the
sensitivity of immunodeficient RAG12/2 mice that lack functional
B and T cells and certain intraepithelial lymphocyte (IEL) subsets
(39) to dsRNA-induced IEC apoptosis. RAG12/2 mice were not
protected from pIC-induced villus shortening and IEC apoptosis
(Supplemental Fig. 4D–F). We also assessed the sensitivity of DCdepleted mice (26) to pIC-activated IEC apoptosis. CD11c-DTR
mice treated with diphtheria toxin to deplete DCs showed similar
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 3. pIC-induced IEC apoptosis is TNF independent. Cleaved caspase 3–stained proximal small
intestine sections from WT mice injected i.p. with
TLR3 ligand pIC (A), TLR7 ligand R848 (B), TLR9
ligand ODN 1826 (C), TLR4 ligand LPS (D), 5 mg
TNF (E), or 1 mg TNF (F) after 2 h. Scale bar, 100 mm.
(G) Cleaved caspase 3–labeled cells per villus in WT
mouse proximal small intestine sections 3 h following
treatment as described in (A)–(F). (H) Serum TNF
levels at 1 h after treatments described in (A)–(F). Data
are mean 6 SD for 4–9 mice/group. **p , 0.01 versus
PBS; other TLR ligands and TNF-treated mice, *p ,
0.05 versus PBS. Cleaved caspase 3–stained proximal
small intestine sections from WT (I) and TNF2/2 (J)
mice at 3 h after i.p. injection of 30 mg/g pIC. Scale
bar, 100 mm. (K) Cleaved caspase 3–labeled cells per
villus in proximal small intestine sections 3 h after 30
mg/g i.p. pIC. (L) Serum TNF levels at 1 h following
i.p. injection of 30 mg/g pIC in WT and TNF2/2 mice.
Values are mean 6 SD, n = 9–14 mice/group.
The Journal of Immunology
423
levels of IEC apoptosis as WT mice in response to pIC (Supplemental Fig. 4K–O). Interestingly, DC-depleted mice had significantly decreased serum TNF levels after pIC treatment (Supplemental
Fig. 4P). DC depletion was confirmed by immunohistochemistry
(Supplemental Fig. 4Q, 4R).
To determine whether other cells of hematopoietic origin contribute to dsRNA-induced IEC apoptosis after pIC, we generated
TLR3 2/2 bone marrow chimera mice. As shown in Fig. 6, WT
mice reconstituted with TLR3-deficient bone marrow (TLR32/2
BM→WT) were as sensitive to pIC-induced IEC apoptosis as WT
mice (Fig 6 A, 6C). In addition, TLR32/2 mice reconstituted with
WT bone marrow (WT BM→TLR32/2) did not show increased
IEC apoptosis in response to pIC (Fig. 6B, 6D). Numbers of
cleaved caspase 3–positive IECs per villus in these mice demonstrated that IEC apoptosis was not dependent on hematopoietic
cells of WT origin (Fig. 6E). Successful bone marrow transfer was
apparent from assaying serum TNF levels in mice after treatment
with pIC (Fig. 6F). WT mice showed TNF levels consistent with
previous experiments and TLR32/2 mice showed no induction of
TNF after pIC, whereas TLR32/2 BM→WT mice had levels of
TNF comparable to TLR32/2 mice and WT BM→TLR32/2 mice
had TNF levels comparable to WT mice (Fig. 6F).
Caspase 8 coordinates IEC apoptosis and preserves the
integrity of the intestinal epithelium following dsRNA
treatment
We observed activated caspase 8 at early time points following pIC
treatment (Supplemental Fig. 1D, 1E). TRIF has been shown to
activate apoptosis through caspase 8, the same initiator caspase that
mediates TNF-induced apoptosis (8, 10). To assess the importance
of caspase 8 in pIC-activated IEC apoptosis and subsequent small
intestinal damage, we generated mice that lack caspase 8 in IECs.
Control mice with floxed caspase 8 (casp8 fl) behaved like WT
mice following pIC treatment and had numerous mid to upper
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 4. IL-15 signaling, NK+ cells, and type I IFN signaling are not required for pIC-induced villus shortening and IEC apoptosis. Cleaved caspase
3–stained proximal small intestine sections from WT (A) and IL-152/2 (B) mice 3 h after 30 mg/g i.p. pIC. Proximal small intestine villus length (C) and
cleaved caspase 3–labeled (D) cells per villus following the treatments in (A) and (B) at 3 h. Cleaved caspase 3–stained proximal small intestine sections for
WT mice treated with Ab isotype control IgG2a (E) or NK1.1 (F) Ab for 24 h prior to 3 h 30 mg/g i.p. pIC treatment. Proximal small intestine villus length
(G) and cleaved caspase 3–labeled (H) cells per villus for mice treated as in (E) and (F). Data are mean 6 SD, n = 5–9 mice/group. (I) IFN-b mRNA levels
normalized to GAPDH using quantitative real-time PCR analysis of WT and TLR32/2 mice treated with PBS or 30 mg/g i.p. pIC. Reverse transcription
used RNA extracted from samples 3 h posttreatment. Data are mean 6 SD of normalized data, n = 3–4 mice/group. **p , 0.01 versus WT PBS and
TLR32/2 pIC. Cleaved caspase 3–stained proximal small intestine sections from WT (J), IFNAR2/2 (K), WT (N), IRF32/2 (O), and BAX2/2 (P) mice 3 h
after 30 mg/g i.p. pIC. Proximal small intestine villus length (L, Q) and cleaved caspase 3–labeled cells per villus (M, R) from these pIC-treated mice at 3 h.
Graphs show mean 6 SD, n = 4–5 mice/group. Scale bar, 100 mm.
424
TLR3–TRIF–CASPASE 8 PATHWAY SIGNALS EPITHELIAL APOPTOSIS
villus IECs that were positive for activated caspase 3 (Fig. 7B,
7C). However, mice lacking caspase 8 in IECs (casp8DIEC) did not
have significant levels of activated caspase 3 in villus IECs indicative of epithelial apoptosis (Fig. 7E). Interestingly, by 2.5 h
posttreatment, these mice manifested destruction of the small intestinal villi, with a loss of the villus epithelium (Fig. 7F) and died
within 6 h of treatment (data not shown). These results demonstrate that caspase 8 is required to regulate IEC apoptosis following
pIC-activated TLR3–TRIF signaling in villus IECs. In the absence
of caspase 8, signaling through TLR3–TRIF causes villus destruction and death.
Discussion
We elucidated the signaling pathways and mechanisms by which
the viral dsRNA mimic pIC selectively causes small intestinal
mucosal damage in vivo. Small intestinal mucosal changes after
encountering pIC were characterized by a rapid onset of epithelial
cell apoptosis, the loss of IECs from villus structures, accompanying marked loss of villus height, and significant diarrhea with
recovery over the following 24–48 h. IEC apoptosis and villus
shortening after pIC treatment were strictly dependent on signaling through the TLR3–TRIF pathway and caspase 8 as demonstrated using TLR32/2, TRIF2/2, and caspase 8DIEC mice. Although
this apoptotic pathway has been studied in vitro (40), we demonstrate an in vivo system where it dramatically impacts the
structure and function of the small intestine.
FIGURE 6. Cells of hematopoietic origin are not required for pIC-induced IEC apoptosis. Cleaved caspase 3 staining of WT (A), TLR32/2 (B),
TLR32/2 bone marrow–reconstituted WT (TLR32/2 BM→WT) (C), and
WT bone marrow–reconstituted TLR32/2 (WT BM→TLR32/2) (D) mice
following 30 mg/g pIC treatment for 3 h. Scale bar, 100 mm. Cleaved
caspase 3–positive cells per villus (E) and TNF cytokine levels (F) for each
group. Graph show results from each mouse. Bar is mean value. *p , 0.01
compared with WT and TLR32/2 BM→WT; #p , 0.01 compared with
WT and WT BM→TLR32/2.
TNF is also well known for its ability to induce IEC apoptosis
(12), but dsRNA-induced IEC apoptosis was completely independent of TNF as well as other cytokines like IL-6 and IL-1.
Moreover, pIC-induced apoptosis of small intestinal villus IECs
in vivo was independent of the TRIF signaling arm of TLR4 and
dsRNA signaling pathways that use the MAVS adaptor molecule
or PKR.
Induction of type I IFN in IECs is an important antiviral
mechanism (41, 42). However, pIC-induced IEC apoptosis in
the villi and villus shortening was independent of this pathway.
Furthermore, although IRF3 can induce apoptosis in response to
viral infection through a BAX-dependent pathway in fibroblast
cell lines (6), we found no role for BAX and IRF3 in pIC-induced
apoptosis of small intestinal IECs.
dsRNA-induced small IEC apoptosis, mucosal remodeling, and
recovery after pIC in vivo required signaling downstream of TLR3–
TRIF through caspase 8. In the absence of an intact signaling
pathway through caspase 8, there was severe damage to the epithelium and small intestinal mucosa. This suggests the caspase 8
apoptotic pathway has an important function as a protective mechanism that maintains the integrity of the intestinal epithelium after
dsRNA-activated TLR3–TRIF signaling. Moreover, the lack of
significant caspase 3 activation in pIC-treated villus IECs from
caspase 8DIEC mice suggests IEC death and loss by an alternative
death pathway such as necroptosis, as seen after TNF treatment
(16, 40, 43).
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 5. pIC- and TNF-induced IEC apoptosis is independent of NFkB signaling, but NF-kB signaling protects crypt cells from apoptosis.
Cleaved caspase 3–stained proximal small intestine sections from IKKa/
b+/+ control (A), IKKa/bDIEC (B), IKKaDIEC (C), and IKKbDIEC (D) mice
at 3 h after 30 mg/g i.p. pIC. Cleaved caspase 3–labeled cells 3 h after 30
mg/g i.p. pIC treatment per villus (E) and per crypt (F). Data are mean 6
SD, n = 3–5 mice/group. *p , 0.05 or **p , 0.01 compared with WT,
IKKa/b+/+, and IKKaDIEC. Cleaved caspase 3–stained proximal small
intestine sections from IKKb+/+ (G) and IKKbDIEC (H) mice 3 h after 5 mg
i.p. TNF. Cleaved caspase 3–labeled cells per villus (I) or per crypt (J) for
IKKb+/+ and IKKbDIEC mice 3 h after 5 mg i.p. TNF. Values are mean 6
SD, n = 3–5 mice/group. Scale bar, 100 mm.
The Journal of Immunology
425
FIGURE 7. Caspase 8 is required for IEC apoptosis and preservation of the epithelium after pIC treatment. Cleaved caspase 3–stained proximal small
intestine sections from Caspase 8fl control mice treated with PBS (A) or 30 mg/g i.p pIC for 1.5 h (B) or 2.5 h (C) and mice lacking caspase 8 in their IECs
(casp8DIEC) treated with PBS (D) or 30 mg/g i.p pIC for 1.5 h (E) or 2.5 h (F). Scale bar, 100 mm.
fluid absorption would culminate in diarrhea that flushes out virus.
Conversely, although TLR32/2 and TRIF2/2 mice are protected
from dsRNA-induced epithelial cell apoptosis, villus remodeling,
and diarrhea, therapeutic approaches designed to inhibit TLR3
signaling could result in decreased virus clearance, as was reported in TLR32/2 mice infected with murine rotavirus (61).
The mechanisms, which we show are responsible for pICinduced small IEC death and villus shortening, differ from those
proposed by Zhou et al. (4, 62). Their model was based on a mix of
in vitro and in vivo experiments in which pIC signaling through
TLR3 was proposed to increase IL-15 production by IECs and
Rae1, a ligand for NKG2D on IECs. Furthermore, IEC-produced
IL-15 was proposed to increase NKG2D on IELs, with coculture
of isolated IECs and IELs, resulting in killing of IECs in vitro at
24 h. Support for their model of pIC-induced small intestinal injury was based on an in vivo finding that Ab to IL-15Ra partially
decreased villus shortening in pIC-injected mice (62). Whereas
dsRNA has been reported to induce IL-15 in vivo (63), using IL152/2 mice we showed that pIC-induced apoptosis and villus
shortening after pIC was totally independent of IL-15. Zhou et al.
(62) further reported decreased small intestinal villus damage in
response to dsRNA after NK1.1 cell depletion, whereas NK1.1
cell depletion did not affect dsRNA-induced IEC apoptosis or
villus shortening in our studies. In addition, using bone marrow
chimera mice we showed that cells of hematopoietic origin were
not required for IEC apoptosis following pIC treatment.
Finally, the relevance of our findings can be envisioned to extend
beyond viral RNA triggers of TLR3–TRIF signaling. In this
regard, mRNA released from necrotic cells was shown to activate
TLR3 signaling (64). Furthermore, RNA from necrotic cells in
rheumatoid arthritis synovial tissues activated TLR3-dependent
signaling (65). In other studies, endogenous RNA from injured
tissues and cells activated TLR3 signaling and resulted in cell
death. For example, endogenous RNA, derived from lung tissue or
necrotic neutrophils, signaled through TLR3 in the absence of
exogenous virus and increased lung damage and death in a model
of hyperoxia lung injury (66). TLR3 also functioned as an endogenous sensor of tissue necrosis, independent of viral activation, in models of septic peritonitis and ischemic gut injury (67).
Importantly, injury and mortality were attenuated in anti-TLR3
Ab treated and TLR32/2 mice. Taken together with our results,
these studies indicate that dsRNA associated with either virus
infection or tissue injury and inflammation can have a dramatic
impact on mucosal tissues.
Acknowledgments
We thank Sandra M. Peterson, Daniel T. Dempsey, Michael R. Wang, and
Kim N. Vu for expert technical assistance. We thank Eyal Raz for providing
IL-1R2/2 mice, Michael David for providing IRF32/2 mice, and Ekihiro
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Cells generated in the stem cell compartment in the crypts of the
small intestine proliferate and migrate upward to populate the villi
where they differentiate and ultimately are shed from the upper
villus tips. Apoptosis of IECs after pIC was largely confined to the
mid to upper villus region. Activation of NF-kB in IECs is important both for the induction of IEC innate proinflammatory
chemokines and cytokines and for IEC resistance to apoptosis (27,
38, 44, 45). Using mice conditionally deficient in IKKa, IKKb, or
both in IECs, we showed that activation of the canonical NF-kB
pathway protected crypt IECs but not villus IECs from pICinduced as well as TNF-induced apoptosis. Moreover, the marked
sensitivity to dsRNA-induced apoptosis of mid to upper villus
IECs in the small intestine occurred in the absence of additional
stimuli. This differs from many other cell types and tissues that
require two stimuli to induce apoptosis (e.g., the activation of
death receptors and translation inhibition) (11). For example, this
is the case in vivo for hepatocytes where both TNF and D-galactosamine, a translation inhibitor, are required to induce apoptosis
(46). Even in certain cancer cells, dsRNA-induced apoptosis
through TLR3 requires a translation inhibitor (47). In those cells,
prosurvival signals from NF-kB signaling appear to protect from
the activation of apoptosis by caspase 8 (11, 48). It is possible that
the different susceptibility of crypt and villus IECs to apoptosis
may reflect differences in the negative regulation of NF-kB signaling in these cells, the state of proliferation or differentiation of
those cells, or different NF-kB isoforms throughout the crypt
villus axis, where an increased fraction of p50 homodimers may
act to negatively regulate NF-kB targets in the villi (38, 49, 50). In
this regard, NF-kB signaling likely is tightly regulated in villus
IECs because these cells ultimately must undergo detachmentdependent apoptosis, termed anoikis, at the villus tips as a part of
their lifecycle and NF-kB signaling has been shown to decrease
anoikis (51).
Infection with enteric viruses like rotavirus causes significant
loss of small intestinal villus epithelial cells, villus shortening, and
concomitant diarrhea (52, 53) similar to that noted in this paper.
Other Reoviridae also contain a dsRNA genome, whereas other
viruses that induce gastroenteritis, including norovirus, produce
dsRNA during their replication cycle (54–56). The likely relevance of the model using parenterally administered synthetic dsRNA
to enteric virus infection is strengthened further because circulating
dsRNA is found in rotavirus-infected humans and animals (57–60).
Our findings with dsRNA suggest a host protective role for the
TLR3–TRIF–caspase 8 activated apoptosis pathway in IECs in
viral infection in vivo. This would result in a rapid shedding of apoptotic virus–infected cells with shortening of villi and a decreased
epithelial surface area available for virus infection. Furthermore,
the generation of fluid in the intestinal lumen by crypt secretory
epithelium concurrent with less epithelial surface available for
426
TLR3–TRIF–CASPASE 8 PATHWAY SIGNALS EPITHELIAL APOPTOSIS
Seki for providing TLR42/2 mice. We also thank Elaine Hanson and Lucia
Hall for maintenance of the mouse colony. We thank Dr. Nissi Varki
for assistance with immunohistochemistry.
Disclosures
25.
26.
The authors have no financial conflicts of interest.
References
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
1. Abreu, M. T. 2010. Toll-like receptor signalling in the intestinal epithelium: how
bacterial recognition shapes intestinal function. Nat. Rev. Immunol. 10: 131–144.
2. Kawai, T., and S. Akira. 2008. Toll-like receptor and RIG-I‑like receptor signaling. Ann. N. Y. Acad. Sci. 1143: 1–20.
3. Thompson, A. J. V., and S. A. Locarnini. 2007. Toll-like receptors, RIG-I‑like
RNA helicases and the antiviral innate immune response. Immunol. Cell Biol.
85: 435–445.
4. Zhou, R., H. Wei, R. Sun, and Z. Tian. 2007. Recognition of double-stranded
RNA by TLR3 induces severe small intestinal injury in mice. J. Immunol. 178:
4548–4556.
5. Clarke, P., and K. L. Tyler. 2009. Apoptosis in animal models of virus-induced
disease. Nat. Rev. Microbiol. 7: 144–155.
6. Chattopadhyay, S., J. T. Marques, M. Yamashita, K. L. Peters, K. Smith,
A. Desai, B. R. Williams, and G. C. Sen. 2010. Viral apoptosis is induced by
IRF-3‑mediated activation of Bax. EMBO J. 29: 1762–1773.
7. Kalai, M., G. Van Loo, T. Vanden Berghe, A. Meeus, W. Burm, X. Saelens, and
P. Vandenabeele. 2002. Tipping the balance between necrosis and apoptosis in
human and murine cells treated with interferon and dsRNA. Cell Death Differ. 9:
981–994.
8. Kaiser, W. J., and M. K. Offermann. 2005. Apoptosis induced by the Toll-like
receptor adaptor TRIF is dependent on its receptor interacting protein homotypic
interaction motif. J. Immunol. 174: 4942–4952.
9. Feoktistova, M., P. Geserick, B. Kellert, D. P. Dimitrova, C. Langlais, M. Hupe,
K. Cain, M. MacFarlane, G. Häcker, and M. Leverkus. 2011. cIAPs block
Ripoptosome formation, a RIP1/caspase-8 containing intracellular cell death
complex differentially regulated by cFLIP isoforms. Mol. Cell 43: 449–463.
10. Estornes, Y., F. Toscano, F. Virard, G. Jacquemin, A. Pierrot, B. Vanbervliet,
M. Bonnin, N. Lalaoui, P. Mercier-Gouy, Y. Pacheco, et al. 2012. dsRNA induces
apoptosis through an atypical death complex associating TLR3 to caspase-8. Cell
Death Differ. 19: 1482‑1494.
11. Van Herreweghe, F., N. Festjens, W. Declercq, and P. Vandenabeele. 2010. Tumor necrosis factor-mediated cell death: to break or to burst, that’s the question.
Cell. Mol. Life Sci. 67: 1567–1579.
12. Piguet, P. F., C. Vesin, J. Guo, Y. Donati, and C. Barazzone. 1998. TNF-induced
enterocyte apoptosis in mice is mediated by the TNF receptor 1 and does not
require p53. Eur. J. Immunol. 28: 3499–3505.
13. Lau, K. S., A. M. Juchheim, K. R. Cavaliere, S. R. Philips, D. A. Lauffenburger,
and K. M. Haigis. 2011. In vivo systems analysis identifies spatial and temporal
aspects of the modulation of TNF-a‑induced apoptosis and proliferation by
MAPKs. Sci. Signal. 4: ra16.
14. Beisner, D. R., I. L. Ch’en, R. V. Kolla, A. Hoffmann, and S. M. Hedrick. 2005.
Cutting edge: innate immunity conferred by B cells is regulated by caspase-8. J.
Immunol. 175: 3469–3473.
15. Madison, B. B., L. Dunbar, X. T. Qiao, K. Braunstein, E. Braunstein, and
D. L. Gumucio. 2002. Cis elements of the villin gene control expression in restricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes
of the intestine. J. Biol. Chem. 277: 33275–33283.
16. Günther, C., E. Martini, N. Wittkopf, K. Amann, B. Weigmann, H. Neumann,
M. J. Waldner, S. M. Hedrick, S. Tenzer, M. F. Neurath, and C. Becker. 2011.
Caspase-8 regulates TNF-a‑induced epithelial necroptosis and terminal ileitis.
Nature 477: 335–339.
17. Hoebe, K., X. Du, P. Georgel, E. Janssen, K. Tabeta, S. O. Kim, J. Goode, P. Lin,
N. Mann, S. Mudd, et al. 2003. Identification of Lps2 as a key transducer of
MyD88-independent TIR signalling. Nature 424: 743–748.
18. Knudson, C. M., K. S. Tung, W. G. Tourtellotte, G. A. Brown, and
S. J. Korsmeyer. 1995. Bax-deficient mice with lymphoid hyperplasia and male
germ cell death. Science 270: 96–99.
19. Mombaerts, P., J. Iacomini, R. S. Johnson, K. Herrup, S. Tonegawa, and
V. E. Papaioannou. 1992. RAG-1‑deficient mice have no mature B and
T lymphocytes. Cell 68: 869–877.
20. Kopf, M., H. Baumann, G. Freer, M. Freudenberg, M. Lamers, T. Kishimoto,
R. Zinkernagel, H. Bluethmann, and G. Köhler. 1994. Impaired immune and
acute-phase responses in interleukin-6‑deficient mice. Nature 368: 339–342.
21. Glaccum, M. B., K. L. Stocking, K. Charrier, J. L. Smith, C. R. Willis,
C. Maliszewski, D. J. Livingston, J. J. Peschon, and P. J. Morrissey. 1997.
Phenotypic and functional characterization of mice that lack the type I receptor
for IL-1. J. Immunol. 159: 3364–3371.
22. Kennedy, M. K., M. Glaccum, S. N. Brown, E. A. Butz, J. L. Viney, M. Embers,
N. Matsuki, K. Charrier, L. Sedger, C. R. Willis, et al. 2000. Reversible defects
in natural killer and memory CD8 T cell lineages in interleukin 15-deficient
mice. J. Exp. Med. 191: 771–780.
23. Van Uden, J. H., C. H. Tran, D. A. Carson, and E. Raz. 2001. Type I interferon is
required to mount an adaptive response to immunostimulatory DNA. Eur. J.
Immunol. 31: 3281–3290.
24. Yang, Y. L., L. F. Reis, J. Pavlovic, A. Aguzzi, R. Schäfer, A. Kumar,
B. R. Williams, M. Aguet, and C. Weissmann. 1995. Deficient signaling in mice
devoid of double-stranded RNA-dependent protein kinase. EMBO J. 14: 6095–
6106.
Honda, K., S. Sakaguchi, C. Nakajima, A. Watanabe, H. Yanai, M. Matsumoto,
T. Ohteki, T. Kaisho, A. Takaoka, S. Akira, et al. 2003. Selective contribution of
IFN-a/b signaling to the maturation of dendritic cells induced by doublestranded RNA or viral infection. Proc. Natl. Acad. Sci. USA 100: 10872–10877.
Abe, K., K. P. Nguyen, S. D. Fine, J.-H. Mo, C. Shen, S. Shenouda, M. Corr,
S. Jung, J. Lee, L. Eckmann, and E. Raz. 2007. Conventional dendritic cells
regulate the outcome of colonic inflammation independently of T cells. Proc.
Natl. Acad. Sci. USA 104: 17022–17027.
Eckmann, L., T. Nebelsiek, A. A. Fingerle, S. M. Dann, J. Mages, R. Lang,
S. Robine, M. F. Kagnoff, R. M. Schmid, M. Karin, et al. 2008. Opposing
functions of IKKb during acute and chronic intestinal inflammation. Proc. Natl.
Acad. Sci. USA 105: 15058–15063.
Nenci, A., C. Becker, A. Wullaert, R. Gareus, G. van Loo, S. Danese, M. Huth,
A. Nikolaev, C. Neufert, B. Madison, et al. 2007. Epithelial NEMO links innate
immunity to chronic intestinal inflammation. Nature 446: 557–561.
Hoshino, K., O. Takeuchi, T. Kawai, H. Sanjo, T. Ogawa, Y. Takeda, K. Takeda,
and S. Akira. 1999. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are
hyporesponsive to lipopolysaccharide—evidence for TLR4 as the Lps gene
product. J. Immunol. 162: 3749–3752.
Sato, M., H. Suemori, N. Hata, M. Asagiri, K. Ogasawara, K. Nakao, T. Nakaya,
M. Katsuki, S. Noguchi, N. Tanaka, and T. Taniguchi. 2000. Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for
IFN-a/b gene induction. Immunity 13: 539–548.
Sun, Q., L. Sun, H. H. Liu, X. Chen, R. B. Seth, J. Forman, and Z. J. Chen. 2006.
The specific and essential role of MAVS in antiviral innate immune responses.
Immunity 24: 633–642.
Maaser, C., M. P. Housley, M. Iimura, J. R. Smith, B. A. Vallance, B. B. Finlay,
J. R. Schreiber, N. M. Varki, M. F. Kagnoff, and L. Eckmann. 2004. Clearance of
Citrobacter rodentium requires B cells but not secretory immunoglobulin A
(IgA) or IgM antibodies. Infect. Immun. 72: 3315–3324.
Gareau, M. G., J. Jury, G. MacQueen, P. M. Sherman, and M. H. Perdue. 2007.
Probiotic treatment of rat pups normalises corticosterone release and ameliorates
colonic dysfunction induced by maternal separation. Gut 56: 1522–1528.
Fan, Y., and A. Bergmann. 2010. The cleaved-caspase-3 antibody is a marker of
caspase-9-like DRONC activity in Drosophila. Cell Death Differ. 17: 534–539.
Scheuner, D., R. Patel, F. Wang, K. Lee, K. Kumar, J. Wu, A. Nilsson, M. Karin,
and R. J. Kaufman. 2006. Double-stranded RNA-dependent protein kinase
phosphorylation of the a-subunit of eukaryotic translation initiation factor 2
mediates apoptosis. J. Biol. Chem. 281: 21458–21468.
Balachandran, S., P. C. Roberts, T. Kipperman, K. N. Bhalla, R. W. Compans,
D. R. Archer, and G. N. Barber. 2000. a/b Interferons potentiate virus-induced
apoptosis through activation of the FADD/caspase-8 death signaling pathway. J.
Virol. 74: 1513–1523.
Jiang, Z., T. W. Mak, G. Sen, and X. Li. 2004. Toll-like receptor 3-mediated
activation of NF-kB and IRF3 diverges at Toll-IL-1 receptor domain-containing
adapter inducing IFN-b. Proc. Natl. Acad. Sci. USA 101: 3533–3538.
Egan, L. J., L. Eckmann, F. R. Greten, S. Chae, Z. W. Li, G. M. Myhre,
S. Robine, M. Karin, and M. F. Kagnoff. 2004. IkB-kinase b-dependent NF-kB
activation provides radioprotection to the intestinal epithelium. Proc. Natl. Acad.
Sci. USA 101: 2452–2457.
Page, S. T., N. S. van Oers, R. M. Perlmutter, A. Weiss, and A. M. Pullen. 1997.
Differential contribution of Lck and Fyn protein tyrosine kinases to
intraepithelial lymphocyte development. Eur. J. Immunol. 27: 554–562.
Mocarski, E. S., J. W. Upton, and W. J. Kaiser. 2012. Viral infection and the
evolution of caspase 8-regulated apoptotic and necrotic death pathways. Nat.
Rev. Immunol. 12: 79–88.
Broquet, A. H., Y. Hirata, C. S. McAllister, and M. F. Kagnoff. 2011. RIG-I/
MDA5/MAVS are required to signal a protective IFN response in rotavirusinfected intestinal epithelium. J. Immunol. 186: 1618–1626.
Hirata, Y., A. H. Broquet, L. Menchén, and M. F. Kagnoff. 2007. Activation of
innate immune defense mechanisms by signaling through RIG-I/IPS-1 in intestinal epithelial cells. J. Immunol. 179: 5425–5432.
Linkermann, A., J. H. Brasen, F. De Zen, R. Weinlich, R. A. Schwendener,
D. R. Green, U. Kunzendorf, and S. Krautwald. 2012. Dichotomy between RIP1and RIP3-mediated necroptosis in tumor necrosis factor a-induced shock. Mol.
Med. 18: 577‑586.
Elewaut, D., J. A. DiDonato, J. M. Kim, F. Truong, L. Eckmann, and
M. F. Kagnoff. 1999. NF-kB is a central regulator of the intestinal epithelial cell
innate immune response induced by infection with enteroinvasive bacteria. J.
Immunol. 163: 1457–1466.
Chae, S., L. Eckmann, Y. Miyamoto, C. Pothoulakis, M. Karin, and
M. F. Kagnoff. 2006. Epithelial cell IkB-kinase b has an important protective
role in Clostridium difficile toxin A-induced mucosal injury. J. Immunol. 177:
1214–1220.
Leist, M., F. Gantner, I. Bohlinger, G. Tiegs, P. G. Germann, and A. Wendel.
1995. Tumor necrosis factor-induced hepatocyte apoptosis precedes liver failure
in experimental murine shock models. Am. J. Pathol. 146: 1220–1234.
Jiang, Q., H. Wei, and Z. Tian. 2008. Poly I:C enhances cycloheximide-induced
apoptosis of tumor cells through TLR3 pathway. BMC Cancer 8: 12.
Plümpe, J., N. P. Malek, C. T. Bock, T. Rakemann, M. P. Manns, and
C. Trautwein. 2000. NF-kB determines between apoptosis and proliferation in
hepatocytes during liver regeneration. Am. J. Physiol. Gastrointest. Liver
Physiol. 278: G173–G183.
Inan, M. S., V. Tolmacheva, Q. S. Wang, D. W. Rosenberg, and C. Giardina.
2000. Transcription factor NF-kB participates in regulation of epithelial cell
The Journal of Immunology
50.
51.
52.
53.
54.
55.
56.
57.
58.
59. Crawford, S. E., D. G. Patel, E. Cheng, Z. Berkova, J. M. Hyser, M. Ciarlet,
M. J. Finegold, M. E. Conner, and M. K. Estes. 2006. Rotavirus viremia and
extraintestinal viral infection in the neonatal rat model. J. Virol. 80: 4820–4832.
60. Moon, S., Y. Wang, P. Dennehy, K. A. Simonsen, J. Zhang, and B. Jiang. 2012.
Antigenemia, RNAemia, and innate immunity in children with acute rotavirus
diarrhea. FEMS Immunol. Med. Microbiol. 64: 382–391.
61. Pott, J., S. Stockinger, N. Torow, A. Smoczek, C. Lindner, G. McInerney,
F. Bäckhed, U. Baumann, O. Pabst, A. Bleich, and M. W. Hornef. 2012. Agedependent TLR3 expression of the intestinal epithelium contributes to rotavirus
susceptibility. PLoS Pathog. 8: e1002670.
62. Zhou, R., H. Wei, R. Sun, J. Zhang, and Z. Tian. 2007. NKG2D recognition
mediates Toll-like receptor 3 signaling-induced breakdown of epithelial homeostasis in the small intestines of mice. Proc. Natl. Acad. Sci. USA 104: 7512–7515.
63. Lodolce, J. P., P. R. Burkett, D. L. Boone, M. Chien, and A. Ma. 2001. T cellindependent interleukin 15Ra signals are required for bystander proliferation. J.
Exp. Med. 194: 1187–1194.
64. Karikó, K., H. Ni, J. Capodici, M. Lamphier, and D. Weissman. 2004. mRNA is
an endogenous ligand for Toll-like receptor 3. J. Biol. Chem. 279: 12542–12550.
65. Brentano, F., O. Schorr, R. E. Gay, S. Gay, and D. Kyburz. 2005. RNA released
from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via Toll-like receptor 3. Arthritis Rheum. 52: 2656–2665.
66. Murray, L. A., D. A. Knight, L. McAlonan, R. Argentieri, A. Joshi, F. Shaheen,
M. Cunningham, L. Alexopolou, R. A. Flavell, R. T. Sarisky, and
C. M. Hogaboam. 2008. Deleterious role of TLR3 during hyperoxia-induced
acute lung injury. Am. J. Respir. Crit. Care Med. 178: 1227–1237.
67. Cavassani, K. A., M. Ishii, H. Wen, M. A. Schaller, P. M. Lincoln, N. W. Lukacs,
C. M. Hogaboam, and S. L. Kunkel. 2008. TLR3 is an endogenous sensor of
tissue necrosis during acute inflammatory events. J. Exp. Med. 205: 2609–2621.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
turnover in the colon. Am. J. Physiol. Gastrointest. Liver Physiol. 279: G1282–
G1291.
Tong, X., L. Yin, R. Washington, D. W. Rosenberg, and C. Giardina. 2004. The
p50-p50 NF-kB complex as a stimulus-specific repressor of gene activation. Mol.
Cell. Biochem. 265: 171–183.
Toruner, M., M. Fernandez-Zapico, J. J. Sha, L. Pham, R. Urrutia, and L. J. Egan.
2006. Antianoikis effect of nuclear factor-kB through up-regulated expression of
osteoprotegerin, BCL-2, and IAP-1. J. Biol. Chem. 281: 8686–8696.
Davidson, G. P., and G. L. Barnes. 1979. Structural and functional abnormalities
of the small intestine in infants and young children with rotavirus enteritis. Acta
Paediatr. Scand. 68: 181–186.
Salim, A. F., A. D. Phillips, J. A. Walker-Smith, and M. J. Farthing. 1995. Sequential changes in small intestinal structure and function during rotavirus infection in neonatal rats. Gut 36: 231–238.
Otto, P. H., I. N. Clarke, P. R. Lambden, O. Salim, J. Reetz, and E. M. LieblerTenorio. 2011. Infection of calves with bovine norovirus GIII.1 strain Jena virus:
an experimental model to study the pathogenesis of norovirus infection. J. Virol.
85: 12013–12021.
Ahlquist, P. 2006. Parallels among positive-strand RNA viruses, reverse-transcribing
viruses and double-stranded RNA viruses. Nat. Rev. Microbiol. 4: 371–382.
McCartney, S. A., L. B. Thackray, L. Gitlin, S. Gilfillan, H. W. Virgin, and
M. Colonna. 2008. MDA-5 recognition of a murine norovirus. PLoS Pathog. 4:
e1000108.
Blutt, S. E., M. Fenaux, K. L. Warfield, H. B. Greenberg, and M. E. Conner.
2006. Active viremia in rotavirus-infected mice. J. Virol. 80: 6702–6705.
Chiappini, E., C. Azzari, M. Moriondo, L. Galli, and M. de Martino. 2005.
Viraemia is a common finding in immunocompetent children with rotavirus
infection. J. Med. Virol. 76: 265–267.
427