Crucial Role of TNF Receptors 1 and 2 in the Control of
Thomas Secher,* Virginie Vasseur,* Didier Marc Poisson,†Jane A. Mitchell,‡
Fernando Q. Cunha,§Jose ´ Carlos Alves-Filho,¶and Bernhard Ryffel2*?
Sepsis is still a major cause of mortality in the intensive critical care unit and results from an overwhelming immune response to
the infection. TNF signaling pathway plays a central role in the activation of innate immunity in response to pathogens. Using a
model of polymicrobial sepsis by i.p. injection of cecal microflora, we demonstrate a critical role of TNFR1 and R2 activation in
the deregulated immune responses and death associated with sepsis. A large and persistent production of TNF was found in
wild-type (B6) mice. TNFR1/R2-deficient mice, compared with B6 mice, survive lethal polymicrobial infection with enhanced
neutrophil recruitment and bacterial clearance in the peritoneal cavity. Absence of TNFR signaling leads to a decreased local and
systemic inflammatory response with diminished organ injury. Furthermore, using TNFR1/R2-deficient mice, TNF was found to
be responsible for a decrease in CXCR2 expression, explaining reduced neutrophil extravasation and migration to the infectious
site, and in neutrophil apoptosis. In line with the clinical experience, administration of Enbrel, a TNF-neutralizing protein, induced
however only a partial protection in B6 mice, with no improvement of clinical settings, suggesting that future TNF immunomodulatory
strategies should target TNFR1 and R2. In conclusion, the present data suggest that the endogenous TNFR1/R2 signaling pathway in
polymicrobial sepsis reduces neutrophil recruitment contributing to mortality and as opposed to pan-TNF blockade is an important
therapeutic target for the treatment of polymicrobial sepsis. The Journal of Immunology, 2009, 182: 7855–7864.
intensive critical care unit (2). The most common causes are severe
pneumonia and intra-abdominal infections, such as peritonitis and
bacteremia, induced by surgical devices (3).
Upon bacterial infection or exposure to a large range of micro-
bial products, two major types of systemic dysfunctions may be the
basis of sepsis. On the one hand, an overwhelming inflammation
may cause a profound suppression of the immune response. On the
other hand, sepsis may induce a robust and overwhelming immune
reaction leading to excessive production of mediators which, by
inducing fever, cardiovascular dysfunction, and multiple organ
failure, are harmful for the host (4). In the investigations of these
complex pathophysiological syndromes, TNF was considered as a
central mediator. Indeed, in vivo injection of TNF partially reca-
pitulate symptoms of septic shock by inducing hypotension, car-
diac dysfunction, and vascular leakage (5).
epsis is generally defined as the result of a systemic in-
flammatory response caused by uncontrolled infection (1).
This is still a major cause of morbidity and mortality in the
In this context, the role of TNF and its closest relative lympho-
toxin ? (LT?)3have been extensively studied. TNF is produced by
many cell types in vivo and it exerts numerous physiological func-
tions by acting on specific receptors (6, 7). Upon cell activation,
TNF is first expressed on the cellular membrane and then cleaved
by the protease TNF-converting enzyme to soluble TNF trimer.
Homotrimeric membrane and soluble TNF or LT mediate similar,
but distinct functions (8–10) through their interaction with two
receptors, TNFR1 (CD120?) and TNFR2 (CD120?). TNF has
both beneficial and deleterious effects during infection. TNF en-
hances leukocytes recruitment and angiogenesis and accelerates
the elimination of various pathogens such as Leishmania (11), Lis-
teria (12), and Mycobacterium (13). In contrast, TNF causes mor-
tality during sepsis and septic shock. TNF inhibitors protect mice
from sepsis induced by LPS or bacteria inoculation (14, 15).
TNFR1 deficiency protects mice from LPS/D-galactosamine shock
as demonstrated using genetically modified mice (16) or soluble
TNFR1 (17). In the colon ascendens stent peritonitis model,
TNFR1-deficient mice present the same mortality rate as the B6
control mice (18). In the cecal ligation and puncture model, mor-
tality is increased after TNF Ab neutralization or in TNFR1-defi-
cient mice (15, 19). In contrast, few studies focused on the role of
TNFR2 in the pathophysiology of sepsis, although a recent report
has indicated that TNFR2 deficiency seems to increase suscepti-
bility to sepsis (20).
After cell stimulation by various stimuli, including TNF-?
itself, these two receptors can be proteolytically cleaved into
two soluble forms, sTNFR1 and sTNFR2, which can be de-
tected at high concentrations and for a prolonged period of time
*Molecular Immunology and Embryology, University of Orleans and Centre National
de la Recherche Scientifique, France;†Regional Hospital of Orleans, Orleans, France;
‡Cardiothoracic Pharmacology, Pharmacology and Toxicology, National Heart and
Lung Institute, Imperial College of London, London, United Kingdom;§Department
of Pharmacology, School of Medicine of Ribeira ˜o Preto, University of Sa ˜o Paulo, Sa ˜o
Paulo, Brazil;¶Division of Immunology, Infection and Inflammation, University of
Glasgow, Glasgow, United Kingdom; and?Institute of Infectious Disease and Mo-
lecular Medicine, University of Cape Town, Republic of South Africa
Received for publication December 2, 2008. Accepted for publication April 9, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the Centre National de la Recherche Scientifique and
the Medical Research Foundation (Fondation pour la Recherche Me ´dicale Francais)
and the European Union (to T.S.).
2Address correspondence and reprint requests to Dr. Bernhard Ryffel, Centre Na-
tional de la Recherche Scientifique, Unite ´ Mixte de Recherche 6218 Molecular Im-
munology and Embryology, Institut de Transge ´nose, 3B rue de la Fe ´rollerie, 45071
Orleans, Cedex 2, France. E-mail address: email@example.com
3Abbreviations used in this paper: LT, lymphotoxin; PLF, peritoneal lavage fluid;
KC, keratinocyte-derived chemokine; MPO, myeloperoxidase; MODS, multiple or-
gan dysfunction syndrome; HTAB, hexadecyltrimethyl ammonium bromide;
sTNFR1/sTNRF2, soluble TNFR1/TNFR2.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
in the circulation of patients with various inflammatory diseases
including LPS-induced sepsis (21, 22). These soluble receptors
are involved in the control of cytokine activity by inhibiting
their ability to bind their membrane receptors and generating a
For the first time and to better understand the role of the
TNF-TNFR axis in polymicrobial sepsis and to understand why
neutralizing TNF therapy has proved relatively unhelpful clin-
ically (23), we investigated the immune and inflammatory re-
sponses in TNFR1/R2 double-deficient mice using a polymi-
crobial model of abdominal infection. We report here that
TNFR1 and R2 deficiency is associated with protection as doc-
umented by neutrophil recruitment, bacterial clearance in the
peritoneal cavity, and subsequent enhanced survival. Further-
more, TNFR deficiency leads to an attenuated hyperinflamma-
tion response associated with abdominal infection which pre-
vents the induction of multiple organ failure and death.
Neutrophils play a key role in the control of infection by lim-
iting bacterial growth (24, 25). In the present study, we show
that TNFR1 and R2 signaling pathways can modulate neutro-
phil homeostasis during bacterial infection by first enhancing
chemotaxis and then by decreasing apoptosis. Finally, by con-
trast to results with TNFR1 deletion, pan-TNF neutralization
using Enbrel, which blocks mouse TNF (26), revealed no in-
creased protection from polymicrobial induced sepsis.
Materials and Methods
Mice and reagents
Adult male C57BL/6 (B6), TNFR1, TNFR2-, TNFR1/R2-, and TNF-defi-
cient mice (obtained from The Jackson Laboratory) used in this study have
been described before (27–29). Animals have been backcrossed at least 10
times on the C57BL/6 background. Animals were ?20–25 g and 10–12
wk old. All mice were bred under specific pathogen-free conditions at the
Transgenose Institute (Centre National de la Recherche Scientifique, Or-
leans, France). The animal experiments complied with the French govern-
ment’s ethical and animal experiment regulations.
Polymicrobial septic peritonitis
Sepsis was induced by i.p. injection of fecal preparation (0.200 ml/25 g
body weight). This was prepared from the cecal contents of uninfected
C57BL/6 mice bred under the same conditions and from the same batch.
Cecal contents were suspended in saline and homogenized with the sterile
disposable homogenization system Dispomix (Medic Tools) and were ali-
quoted and stored in 30% glycerol at ?80°C. Control mice received 200 ?l
of normal saline. In some experiments, mice were treated with 30 or 50
mg/kg of Enbrel or with saline by i.p injection 1 h before and immediately
after the injection of cecal contents.
Identification of bacterial strain
Fecal samples were plated on brain-heart infusion-agar (Difco/BD Pharm-
ingen) or COH-agar (Biome ´rieux) for total bacterial counts, Schaedler-
agar to isolate the anaerobic flora, Pyocyanosel-agar to isolate Pseudomo-
nas aeruginosa, Sabouraud to isolate yeast, Chapman to isolate
Staphylococcus, Drigalski to isolate enterobacteria, and CNA to isolate
enterococcus (Biome ´rieux). Plates were incubated at 37°C and CFU were
observed after 24 h.
Clinical monitoring of mortality
In survival studies, clinical score and rectal temperature were assessed
every hour during the first 8 h after fecal peritonitis induction. Clinical
score listed discernible symptoms such as ruffled fur, hunched posture,
diarrhea, motor impairment, closed eyes, and coma and were each allotted
one point. Rectal temperature was monitored using a rectal device (Physi-
temp Instruments). Mortality was assessed during 7 days.
Organ and blood sampling
Mice were sacrificed at 0, 1, 6, and 24 h after fecal peritonitis induction
and a peritoneal lavage was performed with 3 ml of isotonic saline
solution. Liver, spleen, and lung were harvested and separated into two
parts. Blood was drawn from the lower cava vena. Sera was prepared by
centrifugation at 3000 ? g for 10 min at 4°C and then aliquoted and
stocked at ?80°C.
Peritoneal cellular recruitment
Peritoneal lavage fluid (PLF) was obtained by centrifugation at 400 ? g for
10 min at 4°C. The supernatant (cell-free PLF) was stored at ?80°C for
cytokine analysis. An aliquot of the cell pellets was stained with Turk’s
solution and counted, and 100,000 were cells centrifuged on microscopic
slides (cytospin at 1000 rpm for 10 min at room temperature). Air-dried
preparations were fixed and stained with Diff-Quik (Merz & Dade). Dif-
ferential counts were made under light microscopy. One hundred cells were
counted twice for the determination of the relative percentage of each cell
type present in the PLF.
Peritoneal cells were collected 3 h after fecal peritonitis induction. Cells
were washed once in PBS containing 0.5% BSA (PBS/BSA) and stained on
ice at 106cells/100 ?l with primary Abs: anti-GR-1 FITC or PE (clone
1A8), anti-CD11b PerCP (clone M1/70), anti-F4/80 PE (clone BM8), anti-
Ly6C FITC (clone AL-21), and anti-CXCR2 (clone 242216) for 20 min in
the dark. For the apoptosis assay, we used the Annexin V-FITC Apoptosis
Detection Kit I. All Abs were from BD Pharmingen except CXCR2 Ab
(R&D Systems). After washing with PBS/0.5% BSA, cells were analyzed
on a BD Biosciences LSR I.
Cytokines and chemokines determination
TNF, IL-1?, IL-6, keratinocyte-derived chemokine (KC), and MCP-1 in
PLF and whole blood sera were measured by ELISA (Duoset Kit; R&D
Systems) according to the manufacturer’s instructions (with detection lim-
its at 50 pg/ml).
Bacterial organ load
Ten-fold serial dilutions of PLF were plated on brain-heart infusion agar
plates (Biovalley). Plates were incubated at 37°C and 5% CO2, and the
numbers of CFU were enumerated after 24 h. Organ homogenates were
prepared in 3 ml of isotonic saline solution using a Dispomix tissue ho-
mogenizer (Medic Tools).
Myeloperoxidase (MPO) activity
Liver homogenates were centrifuged at 10,000 ? g for 10 min at 4°C and
the supernatant was discarded. The pellets were resuspended in 1 ml of
PBS containing 0.5% hexadecyltrimethyl ammonium bromide (HTAB)
and 5 mM EDTA and then incubated for 2 h at 60°C to inactivate the
endogenous catalases. PLF aliquots were pelleted and resuspended in 1 ml
of PBS-HTAB-EDTA. Following a new centrifugation, 50 ?l of superna-
tant was placed in test tubes with 200 ?l of PBS-HTAB-EDTA, 1.6 ml of
HBSS, 100 ?l of o-dianisidine dihydrochloride (1.25 mg/ml), and 100 ?l
of 0.05% H2O2After 15 min of incubation at 37°C under agitation, the
reaction was stopped with 100 ?l of 1% NaN3. The MPO activity was
determined as absorbance at 460 nm against blanck (reaction mixture with
saline in place of sample).
Liver, spleen, and lung were fixed in 10% buffered formalin (Shandon).
Tissues were dehydrated in ethanol and embedded in paraffin. Sections
(3-?m thick) were stained with H&E for evaluation of pathological
changes by two independent observers. All liver sections were graded as
follows: grade 0, normal histomorphology; grade 1, minor inflammatory
infiltrates with occasional liver cell necrosis; grade 2, moderate liver dam-
age with inflammatory infiltrates and focal necroses; and grade 3, extensive
infiltrates accompanied by diffusely distributed liver cell necroses. At least
two separate sections were assessed per liver.
Preparation of bone marrow neutrophils
Murine bone marrow cells were isolated from femurs and tibiae in 2 ml of
HBSS without Ca2?and Mg2?and laid on top of a two-layer Percoll
gradient of 72 and 65% Percoll (Sigma-Aldrich) diluted in HBSS (100%
Percoll was obtained by mixing nine parts of Percoll and one part of 10?
HBSS) and centrifuged at 1200 ? g for 30 min at room temperature with-
out brake. The enriched neutrophil fraction was recovered at the interface
between 65 and 72% Percoll. After washing twice with HBSS, 5 ? 10?6
cells were obtained per mouse containing 95% of Gr-1-positive cells.
7856ROLE OF TNF IN POLYMICROBIAL SEPSIS
To examine the neutrophil chemoattractant response to MIP-2, a modified
Boyden chamber assay was performed using a 48-well microchamber
(NeuroProbe). Murine bone marrow neutrophils were isolated as above and
resuspended in running buffer (HBSS 1? supplemented with 2 mg/ml
BSA, 10 mM HEPES, 1 mM CaCl2, and 1 mM MgCl2). Recombinant
mouse MIP-2 (30 ng/ml) diluted in running buffer (for wells containing
neutrophils) or appropriate buffer control was added to the lower chambers
of the apparatus. A 5-?m pore polycarbonate membrane (NeuroProbe) was
placed between the upper and lower chambers, and 5 ? 10?4cells in a
volume of 50 ?l were added to the top chambers of the apparatus. Cells
were allowed to migrate into the membrane for 1 h at 37°C with 5% CO2.
Following incubation, the chamber was disassembled and the membrane
was scraped and washed three times in PBS to remove nonadherent cells
before being fixed and stained using Diff-Quik (Merz & Dade). Each well-
associated membrane area was scored using light microscopy to count the
intact cells present in five random fields. The results are expressed as the
number of neutrophils per field.
cient mice are partially protected
from lethal polymicrobial infection.
B6, TNFR1-, and TNF-deficient mice
received injections in the peritoneum
of 200 ?l of cecal contents (3.5 ? 105
CFU). All preparations consist in a
polymorph population of bacteria
composed of Gram-positive and -neg-
ative, aerobic, and anaerobic strains
(A). The concentrations of TNF in se-
rum (B) and PLF (C) in B6 mice 1, 6,
and 24 h after infection were deter-
mined by ELISA. Rectal temperature
(D) and survival rates (E) were mon-
itored during 7 days after the infec-
tion. Groups of six mice were used
and mean values ? SD are shown.
The results are representative of two
TNFR1- or TNF-defi-
100, and 50 ?l of cecal contents corresponding to 3.5 ? 105, 2.25 ? 105, 1.75 ? 105, and 1.35 ? 105CFU. Clinical score (A), rectal temperature (B), and
survival rates (C) were monitored during 7 days after the infection. Groups of six mice were used and mean values ? SD are shown. The results are
representative of four independent experiments.
Dose-response effect of microbial inoculum on survival and clinical parameters. B6 mice were injected in the peritoneum with 200, 150,
7857The Journal of Immunology
Statistical evaluation of differences between the experimental groups was
determined by using the log rank test for survival curves and both Student’s
t test (comparing two groups) and one-way ANOVA followed by a Bon-
ferroni post test (comparing more than two groups) for others data. All tests
were performed with GraphPad Prism. All data are presented as mean ?
SD. A p ? 0.05 was considered significant: ?, p ? 0.05; ??, p ? 0.01; and
???, p ? 0.001.
TNF or TNFR1 deficiency improves clinical parameters and
survival in polymicrobial peritonitis
Polymicrobial peritonitis and sepsis result in enhanced release of
TNF and other proinflammatory cytokines causing a hyperin-
flammatory syndrome (30). To study the implication of the TNF
pathway in polymicrobial sepsis-induced immune responses,
we injected i.p. bacteria obtained from the cecum of control B6
mice. The inoculum represents mixed intestinal microbiota
composed of aerobic and aero/anaerobic microbes and the main
strains identified were Escherichia coli, Enterococcus, Staphy-
lococcus, and Lactobacillus sp. (Fig. 1A). First, we established
a dose-response effect with the standard preparation of fecal
preparation, which causes a dose-dependent hypothermia and
death (Fig. 2).
The injection of 3.5 ? 105CFU of these intestinal microbes
induced a rapid local and systemic production of TNF which was
maximal at 6 h (Fig. 1B) and correlates with increased TNF serum
levels (Fig. 1C). Thus, polymicrobial sepsis leads to a marked local
and systemic production of TNF.
Using TNF- or TNFR1-deficient mice, we asked whether
TNF contributes to the development of the clinical signs of
in the peritoneum of 200 ?l of cecal contents (3.5 ? 105CFU). Clinical score (A), rectal temperature (B), and survival rates (C) were monitored during
7 days after the infection. Groups of six mice were used and mean values ? SD are shown. The results are representative of three independent experiments.
TNFR1/R2-deficient mice are totally protected from lethal polymicrobial infection. Both B6 and TNFR1/2-deficient mice received injection
recruitment in the peritoneum and
bacterial clearance in the absence of
TNFR1/R2. Peritoneal exudates were
harvested from B6 and TNFR1/R2?-
deficient mice 1, 6, and 24 h after the
infection with 200 ?l of cecal con-
tents (3.5 ? 105CFU). Absolute
numbers of neutrophils (A) in PLF
were measured. MPO levels were de-
termined in PLF pellets (B) to assess
neutrophils activation. Peritoneal cell
phenotypes were characterized 3 h af-
ter infection by staining with Gr-1,
F4/80, CD11b, and Ly6C Abs. C,
Representative dot plot. R1 defined
resident macrophages as F4/80???,
Gr-1?, Ly6C??, and CD11b???
CD11b??cells; and R3 defined neu-
Ly6C???, and CD11b??cells. Sub-
sequently, CFU numbers in PLF were
quantified 24 h after the infection (D).
Groups of six mice were used and
mean values ? SD are shown. The
results are representative of three in-
7858 ROLE OF TNF IN POLYMICROBIAL SEPSIS
septic peritonitis. We observed reduced hypothermia in
TNFR1- but not in TNF-deficient mice compared with feces-
injected B6 mice (Fig. 1D). These symptoms appear early after
infection in B6 mice, predicting a poor prognosis (31). Thus,
mice lacking the TNFR1 or the TNF gene are almost fully pro-
tected and had a survival rate around 70% after 7 days, whereas
all B6 mice died within 2 days (Fig. 1E). Therefore, these data
suggested that TNF contributes to the negative outcome of
acute polymicrobial septic peritonitis and that its incomplete
inhibition will lead to a partial protection.
TNFR1/2 deficiency induced total protection in polymicrobial
Following a peritoneal injection of 3.5 ? 105CFU of intestinal
microbes, TNFR1/R2 double-deficient mice were protected from
clinical symptoms, which include reduced locomotion, diarrhea,
and hunched posture (Fig. 3A). Moreover, we observed reduced
hypothermia in TNFR1/R2- deficient mice compared with B6 mice
(Fig. 3B). By contrast to results with TNFR1-only or TNF-defi-
cient mice, mice double deficient in both TNFR1 and R2 were
totally protected with 100% survival of polymicrobial sepsis at 7
days (Fig. 3C). However, absence of TNFR2 unlike the combined
absence of TNFR1 and R2 did not confer protection in this model
of infection (data not shown). Together, these data showed that a
complete abolition of the TNF-dependent signaling pathway can
protect mice from an experimental polymicrobial sepsis.
Enhanced neutrophil recruitment in the peritoneum and
bacterial clearance in the absence of TNFR1/R2
Protection from polymicrobial sepsis and shock is associated with
enhanced cell recruitment into the peritoneal cavity. Neutrophils
and macrophages are the two main cell subtypes present in peri-
toneum after polymicrobial infection which are rapidly recruited
and mediate the initial local inflammatory response (32). TNFR1/
R2-deficient mice showed significantly higher numbers of neutro-
phils (Fig. 4A) and macrophages (data not shown) in PLF, com-
pared with B6 mice, upon infection. Both recruitment and
neutrophil activation can be correlated with the quantification of
the MPO activity (33). In fact, we observed that peritoneal MPO
activity was significantly increased in TNFR1/R2-deficient mice
compared with the B6 mice (Fig. 4B).
Flow cytometric analysis of the inflammatory cells from PLF at
3 h after infection confirmed a dramatic increase of neutrophil
recruitment in TNFR1/R2-deficient mice compared with B6 mice
IL-1?, KC, and MCP-1 in PLF (A) and serum (B) were determined in B6 and TNFR1/R2?-deficient mice 24 h after infection. Cytokines and chemokines
concentrations were determined by ELISA. Groups of six mice were used and mean values ? SD are shown. The results are representative of two
Reduced local and systemic hyperinflammation upon polymicrobial sepsis in the absence of TNF signaling. The concentrations of IL-6,
7859 The Journal of Immunology
upon infection. Resident macrophages were diminished in B6 and
absent in TNFR1/R2-deficient mice and inflammatory monocytes
were increased upon infection in both groups (Fig. 4C). Thus, neu-
trophil recruitment is clearly increased in the absence of TNF
Finally, the analysis of the local bacterial load in PLF showed
that TNFR1/R2-deficient mice had significantly less viable bacte-
ria at 24 h after infection than control B6 mice (Fig. 4D). More-
over, cytospin preparations from the PLF at 24 h showed that
neutrophils and macrophages from TNFR1/R2-deficient mice con-
tained very few bacteria, whereas the B6 controls showed clearly
detectable bacteria suggesting enhanced bacterial killing in the ab-
sence of TNF signaling (data not shown). Therefore, the innate
host immune response to a polymicrobial infection is enhanced
with more effective control of bacterial growth in the absence of
TNFR1/2 deficiency reduces local and systemic cytokine and
chemokine production upon polymicrobial sepsis
TNF and also numerous cytokines are augmented in sepsis. They
are known to activate cellular defense against infection while ex-
aggerated cytokine production may lead to death (34, 35). There-
fore, we investigated the local and systemic levels of inflammatory
mediators upon polymicrobial infection in serum and PLF at 24 h
after infection. This time point was chosen because TNF serum
levels were still high. IL-6, IL-1?, KC, and MCP-1 in PLF (Fig.
5A) and in serum (Fig. 5B) were significantly lower in the absence
of TNFR1/R2 compared with B6 mice. The overall increase of
single mediators was not as dramatic as after endotoxin adminis-
tration. However, the combined action of these mediators might
cause hyperinflammation and tissue damage.
Collectively, these data indicate that cytokines and chemokines
induced by polymicrobial sepsis, are in part dependent on TNF
with reduced production in TNFR1/R2-deficient mice.
Reduced organ injury in the absence of TNFR1/R2
Microscopic examinations of the liver from B6 mice revealed focal
inflammation with single-cell necrosis, focal neutrophil recruit-
ment, and thrombotic lesions in the portal areas (Fig. 6A). These
hepatic changes, especially single-cell necrosis, were less pro-
nounced in TNFR1/R2-deficient mice. The spleen was enlarged
and showed congestion in the red pulp with lymphocyte depletion
in the white pulp in B6 mice. These parameters were reduced in
TNFR1/R2-deficient mice (Fig. 6B). Lungs of B6 mice revealed a
distinct alveolar congestion with cellular infiltrates around bronchi
and capillaries and microthrombi in small vessels, while TNFR1/
R2-deficient mice showed only minimal changes (Fig. 6C).
The hepatic necrosis and neutrophil recruitment in the liver
were assessed semiquantitatively. This analysis confirmed a
significant protection from liver necrosis and inflammation in
the absence of TNF signaling (Fig. 6D), which correlated with
the macroscopic changes. Since neutrophils contribute to liver
injury, we quantified neutrophils by assessing MPO activity,
which was augmented in B6 mice and significantly reduced in
TNFR1/R2-deficient mice (Fig. 6E). Therefore, the complete
absence of TNF signaling reduced multiorgan failure associated
with polymicrobial sepsis.
Enhancement of neutrophil migration and decreased apoptosis
in the absence TNFR1/2 signaling
Reduction of neutrophil migration into the infectious focus during
severe sepsis correlates with the severity of disease (36). This phe-
nomenon may be the consequence of down-regulation of chemo-
kine receptor CXCR2 on the surface of circulating neutrophils
(37). Results in Fig. 3B revealed that TNFR1/R2-deficient mice
display increased neutrophil migration into the site of infection,
suggesting that TNFR1/2 signaling might be involved in failure of
neutrophil migration during severe sepsis. To test whether TNF
negatively regulates neutrophil chemotaxis upon stimulation with
CXCR2 ligand chemokines, we stimulated polymorphonuclear
neutrophils from B6 mice with TNF at different concentrations and
quantified the chemotaxis in response to MIP-2 in a modified Boy-
den chamber. TNF preincubation for 1 h diminished markedly neu-
trophil chemotaxis compared with the medium-treated cells. This
reduction is similar to that induced by LPS (Fig. 7A). Consistent
in the peritoneum of 200 ?l of cecal contents (3.5 ? 105CFU). Liver, lung, and spleen tissues were histologically examined 24 h after infection. Liver
sections (A) showed reduced areas of necrosis and inflammation (dotted arrows show cellular infiltrates, solid arrows show necrotic areas) in TNFR1/
R2-deficient mice. Increased liver injury in B6 compared with TNFR1/R2?-deficient mice (D) with associated elevation of MPO (E). Lung sections
revealed diminished neutrophil recruitment and hemorrhage (dotted arrows show thrombotic foci, solid arrows show cellular infiltrates) in TNFR1/R2-
deficient mice (B). Spleen sections showed minimal congestion and necrosis (dotted arrows show necrotic areas in the white pulp, solid arrows shows
thrombotic congestion in the red/white pulp) in TNFR1/R2-deficient mice (C). All sections were stained with H&E staining and observed at ?20
magnification. Groups of six mice were used and mean values ? SD are shown. The results are representative of three independent experiments.
Reduced organ injury in the absence of TNFR1/R2 upon polymicrobial infection. Both B6 and TNFR1/R2-deficient mice received injections
7860ROLE OF TNF IN POLYMICROBIAL SEPSIS
with these first findings, we found that the chemotaxis of neutro-
phils from TNFR1-deficient mice was not down-regulated by LPS
or lipoteichoic acid (Fig. 7B), confirming that TNF leads to a
marked reduction of neutrophil migration.
To further explore the role of TNF in neutrophil migration fail-
ure, we examined ex vivo the expression of CXCR2 on mice sub-
jected to polymicrobial sepsis. Flow cytometric analysis of neu-
trophils (defined as Gr-1highCD11bhigh) showed that the absence of
TNFR1/2 signaling significantly attenuated the reduction of
CXCR2 expression observed in B6 neutrophils (Fig. 7C).
TNF has a dual effect in neutrophil apoptosis. At low doses and
coupled with others inflammatory stimuli, it prolongs the life span
of circulating neutrophils. By contrast, a high dose and protracted
exposure of TNF accelerates apoptosis (38). To test whether TNF
will induce neutrophil apoptosis during polymicrobial sepsis, we
measured ex vivo staining profiles for annexin V and propidium
iodide (Fig. 7D). We defined annexin V?and propidium iodide?
as early apoptosis (R1), annexin V?and propidium iodide medium
as late apoptosis (R2), and annexin Vhighpropidium iodidehighas
necrosis (R3). We observed that neutrophils from TNFR1/R2- de-
ficient mice exhibit increased early apoptosis compared with the
B6 controls. By contrast, neutrophils from B6 mice exhibited more
late apoptosis (Fig. 7E).
TNF neutralization only partially reduces polymicrobial sepsis
Since our investigations suggest that endogenous TNF may be a
critical pathogenic factor of polymicrobial sepsis, we tested
whether TNF neutralization with a soluble TNFR2 (Enbrel) mod-
ulates the outcome of polymicrobial inflammation. Mice were pre-
treated (?1 and 0 h) with Enbrel (30 mg/kg) or saline and then
inoculated i.p. with the cecal contents. Enbrel slightly reduced the
clinical symptoms of sepsis (Fig. 8A) and hypothermia (Fig. 8B)
and partially enhanced survival (Fig. 8C). Additional experiments
with a higher dose of Enbrel (50 mg/kg) did not improve protec-
tion (data not shown). Furthermore, delayed injection (6 h after the
infection) is ineffective since all of the treated mice died within 2
days (data not shown). Collectively, these data strongly suggest
that complete and early TNF neutralization may be required to
confer protection after sepsis onset.
bone marrow neutrophils from B6 mice and TNFR1-deficient mice were incubated with TNF (10, 50, and 100 ng/ml) (A), LPS (100 ng/ml), or lipoteichoic
acid (LTA; 100 ng/ml) (B) and the chemotaxis induced by MIP-2 was visualized as described in Materials and Methods. Blood neutrophils from infected
and uninfected mice were harvested 3 h after infection and CXCR2 expression was determined by flow cytometry. C, Mean of fluorescence intensity of
CXCR2 expression from five B6 and TNFR1/R2-infected mice. Peritoneal cells from infected mice were harvested 3 h after infection and apoptotic or
necrotic neutrophils were determined by staining with annexin V and propidium iodide. D, Representative dot plot gated on Gr-1?cells. R1 defined early
apoptosis, R2 defined late apoptosis, and R3 defined necrosis. E, Bar graph representing the percentage of Gr-1-positive cells in apoptosis. Groups of five
mice were used and mean values ? SD are shown. The results are representative of two independent experiments.
TNF induce a reduction of neutrophil CXCR2-dependent chemotaxis and CXCR2 expression and an enhancement of apoptosis. Purified
7861 The Journal of Immunology
This is the first report to demonstrate that combined loss of TNFR1
and R2 signaling pathways participate critically in the pathogen-
esis of lethal polymicrobial sepsis while TNF neutralization or
single gene deletion does not provide the same level of protection.
To improve the understanding of the role of the TNF-TNFR axis
in the pathophysiology encountered in sepsis, we used a standard-
ized and highly reproducible murine model of polymicrobial sepsis
also known as fecal peritonitis. We injected i.p. a mixed population
of Gram-positive and -negative, aerobic, and anaerobic commensal
bacteria. Using this model of polymicrobial peritonitis resulting in
sepsis, we revealed that the host responds with a vigorous innate
immune response with the release of proinflammatory cytokines,
including TNF, and chemokines leading to monocyte and neutro-
phil recruitment. We further showed enhanced recruitment of im-
mune cells in the absence of TNFR1/2. Control of the bacterial
growth was enhanced, correlating with a total protection of
TNFR1/R2- deficient mice compared with B6.
Then, we demonstrated that in the absence of TNFR1/R2, the
late hyperinflammatory state was less pronounced than in the B6
controls. Systemic and local levels of IL-6 and IL-1? were re-
duced, which is consistent with the role of TNF on the production
of these cytokines and with the fact that peritoneal sepsis induces
a strong and rapid induction of proinflammatory mediators (39,
40). In addition, chemokine production was also dampened in
TNFR1/R2-deficient mice. KC, the murine homolog of IL-8 which
is known to be a poor prognosis marker in septic patients (41), was
decreased during the late phase of septic peritonitis in TNFR1/R2-
deficient mice while it was equivalent during the early part of the
disease in both TNFR1/R2-deficient and B6 mice (data not
shown). These findings were confirmed with MCP-1 which is also
decreased when TNFR1 and R2 signaling is absent. These chemo-
kines are known to induce the recruitment of neutrophils, partic-
ularly in septic peritonitis (42), but their accumulation will also
lead to uncontrolled inflammation, organ failure, and lethal septic
shock. Thus, our data highlighted the crucial role of TNFR1- and
R2-mediated signaling pathways in the late hyperinflammation in-
duced by sepsis, which is a poor prognosis for the host (43). These
data were similar to those observed in IFN?-R1-deficient mice
undergoing polymicrobial sepsis which identified type I IFN as a
critical inducer of the late production of TNF, KC, and IP-10 (44).
Furthermore, the present study showed that TNFR1/R2-deficient
mice can maintain body temperature during the course of infection
unlike B6 controls. This finding is in accordance with systemic
organ dysfunctions that target liver, spleen, and lung during
polymicrobial sepsis (45). Moreover, we observed that TNFR1/
R2-deficient mice presented reduced central organ injuries com-
pared with B6 controls. Multiple organ dysfunction syndrome
(MODS) has been early defined as the main cause of morbidity in
septic patient (46). It has been correlated with a loss of homeosta-
sis in several interdependent organ systems including loss of con-
trol in body temperature. Septic patients who develop hypothermia
have a significantly worse prognosis compared with those who
have fever or maintain body temperature. In animal models, hy-
pothermia is generally associated with immune dysfunction and
poor outcome (47). Thus, TNFR1/R2-deficient mice were pro-
tected against systemic organ dysfunction which, in addition with
a better bacterial clearance, will correlate the improved survival.
Massive neutrophil recruitment to the site of infection is an es-
sential mechanism to control invading pathogens, especially in in-
tra-abdominal bacterial infection (24, 48). In the present study,
increased neutrophil accumulation in the peritoneal compartment
was correlated with a better control of the bacteria. Thus, this
mechanism may contribute to improved survival of TNFR1/R2-
deficient mice. Chemokines are potent and specific chemoattrac-
tants for polymorphonuclear cells (49). The CXC family comprises
several proteins such as MIP-2 or IP-10 that have been known for
years to have a critical role in humans and animal models of dis-
ease (50–52). CXCR2 has been shown to mediate the responses to
CXC chemokines in polymorphonuclear neutrophils (53). Under
pathological conditions, surface expression of CXCR2 is down-
regulated by 50% on neutrophils from septic patients (54, 55).
Recent studies have shown that a proinflammatory environment
the peritoneum of 200 ?l of cecal contents (3.5 ? 105CFU). Enbrel (30 mg/kg) was given i.p. 1 h before and concomitantly with the inoculum. TNF
neutralization in B6 mice leads to a reduced loss of rectal temperature (A), a diminished severity of clinical signs (B), and an enhanced survival (C). Fourteen
mice were used and mean values ? SD are shown. The results are a pool from two independent experiments.
Neutralization of TNF failed to prevent lethality from polymicrobial sepsis. Both B6- and TNFR1/R2-deficient mice received injections in
7862ROLE OF TNF IN POLYMICROBIAL SEPSIS
can lead to a failure in neutrophil recruitment to the site of infec-
tion (37, 56). In the present study, we also demonstrated that TNF
plays a role in CXCR2 down-regulation which leads to the failure
of the neutrophils to migrate to the site of infection, as the neu-
trophils from TNFR1/R2-deficient exhibited an increased CXCR2
expression compared with the B6 controls.
One of the major pathways in limiting the inflammatory re-
sponse is the clearance of neutrophils and their potentially cyto-
toxic content. Indeed, large numbers of apoptotic neutrophils or
engulfed neutrophils in macrophages have been found in septic
peritonitis (57) or in acute lung inflammation (58). Deregulation of
immune cell apoptosis may be a component of the immune dys-
function and multiple organ failure that occur in sepsis (59, 60).
Indeed, in sepsis, apoptosis inhibition protects animals from le-
thality (61). We therefore asked whether polymicrobial sepsis will
modulate neutrophil accumulation by changing their ability to
progress into apoptosis. The increased level of early apoptosis in
TNFR1/R2-deficient mice may enhance bacterial and immune
clearance and limit the inflammatory response. Furthermore, the
decreased level of late apoptosis and, at the end, necrosis will
prevent the generation of hazardous inflammatory components be-
cause B6 mice seem to exhibit more necrotic cells than TNFR1/R2
later after infection (data not shown).
Finally, the experiments with Enbrel revealed that this treatment
partially protected mice from death without reducing temperature
loss and clinical symptoms appearance. These findings are in ac-
cordance with the results obtained on TNFR1- or TNF-deficient
mice that were only partially protected against the infection. These
results can be explained by the fact that in TNF-deficient mice,
LT?-dependent activation of the TNFR1/2 is present. Then, in
TNFR1 single-deficient mice, TNF or LT?- dependent activation
of TNFR2 is also effective. This would explain why the sole ab-
sence of TNFR2 is not protective. Finally, in both TNF- and
TNFR1-deficient mice, the biological effect of sTNFR1 or
sTNFR2, respectively, is still active. Moreover, a positive corre-
lation exists between TNF-soluble receptors and simultaneously
obtained sepsis score (62). Enbrel-treated mice submitted to an
LPS shock only showed a partial cardiac function restoration and
survival (63). Many studies in septic patients showed that TNF or
IL-1 neutralization did not provide conclusive evidence of improv-
ing the outcome of clinical settings. Only one study has identified
a small subset of critically septic patients that may benefit from
TNF blockade, while in most others studies, no benefit was re-
ported (64). In the context of a sepsis syndrome, our data suggest
that an effective therapeutic neutralization of TNF effects should
target both the TNFR1 and R2 signaling pathway and be initiated
as soon as possible after the onset of the infection.
In conclusion, we demonstrate that the combined activation of
TNFR1 and R2 signaling pathways play a detrimental role in an
experimental model of polymicrobial sepsis. Moreover, the direct
effect of TNF and the associated signaling pathways on neutrophil
homeostasis during an ongoing infection was established and cor-
related with the induction of a delayed immune response and a
hyperflammatory state which will lead to death. Blockade of both
TNFR1/R2 may be required to confer substantial protection of
We gladly thank Prof. Mauro Teixera and Prof. Franc ¸ois Erard for assis-
tance in manuscript preparation.
The authors have no financial conflict of interest.
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