TIMI-782; No.
of Pages 11
Review
Cecal ligation and puncture:
the gold standard model for
polymicrobial sepsis?
Lien Dejager1,2, Iris Pinheiro1,2, Eline Dejonckheere1,2 and Claude Libert1,2
1
Department for Molecular Biomedical Research, Flanders Institute for Biotechnology (VIB), B9052 Ghent, Belgium
2
Department of Biomedical Molecular Biology, Ghent University, B9052 Ghent, Belgium
Sepsis is a serious medical condition characterized by
dysregulated systemic inflammatory responses fol-
lowed by immunosuppression. To study the pathophys-
iology of sepsis, diverse animal models have been
developed. Polymicrobial sepsis induced by cecal liga-
tion and puncture (CLP) is the most frequently used
model because it closely resembles the progression
and characteristics of human sepsis. Here we summarize
the role of several immune components in the patho-
genesis of sepsis induced by CLP. However, several
therapies proposed on the basis of promising results
obtained by CLP could not be translated to the clinic.
This demonstrates that experimental sepsis models do
not completely mimic human sepsis. We propose several
strategies to narrow the gap between experimental
sepsis models and clinical sepsis, including targeting
factors that contribute to the immunosuppressive phase
of sepsis, and reproducing the heterogeneity of human
patients.
Sepsis therapies: search for a needle in a haystack
Tight regulation of the immune system is essential for
maintaining the balance between protective and tissue-
damaging inflammatory responses. Therefore, local in-
flammatory responses are finely regulated and cease when
the causative factor is removed. However, if the inflamma-
tory reaction becomes dysregulated, systemic activation of
the innate immune system becomes excessive and results
in systemic inflammatory response syndrome (SIRS) or
sepsis [1].
SIRS and sepsis are systemic reactions to inflammatory
triggers including infections and sterile causes such as
burns and trauma. SIRS and sepsis are characterized by
the presence of the following clinical manifestations: hy-
perthermia or hypothermia, tachycardia, tachypnea, and
leukocytosis or leukocytopenia (Figure 1) [1]. Although
bacterial infections are the leading cause of sepsis, fungi,
parasites and viruses can also cause sepsis, but less fre-
quently [2]. Blood culture diagnosis of infection is the
current standard for sepsis. However, positive cultures
can be detected in only 20% of sepsis patients. Therefore,
research is currently directed towards finding reliable
biomarkers and molecular diagnostic tests which could
Corresponding author: Libert, C. (Claude.Libert@dmbr.UGent.be).
0966-842X/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2011.01.001 Trends in Microbiology xx (2011) 1–11
improve diagnosis of sepsis and subsequent treatment
[3,4].
Epidemiological records from North America show an
incidence of sepsis of 3 cases per 1000 persons annually, and
this is expected to increase at a rate of 1.5% per year [5].
Despite advances in supportive care, sepsis remains associ-
ated with very high mortality rates (40–60%) and is the
leading cause of death in noncoronary intensive care units
[5]. These data show that sepsis is a huge burden for
healthcare systems and a major challenge in contemporary
medicine.
Current management of sepsis is largely supportive and
consists of administration of broad-spectrum antibiotics,
hemodynamic resuscitation and appropriate support of or-
gan function. These treatments have remained essentially
unchanged for the past three decades [4]. Despite extensive
research and more than 30 phase III randomized trials, only
few new therapies were found to be relatively beneficial [6].
Sepsis is a complex, dynamic syndrome with wide het-
erogeneity among patients, which makes it an important
medical and economic challenge for scientists and clinicians.
The high prevalence, high mortality rates and lack of effec-
tive treatments result in substantial economic costs and
underscore the need for further research. A better under-
standing of the fundamental processes involved in the com-
plex pathology of sepsis is essential for identifying new
therapeutic targets. Different experimental models have
been developed, but which of these is the most representa-
tive of human sepsis is often debated. In this review we
summarize the advantages, disadvantages and limitations
of the most frequently used models of experimental sepsis, of
which none mimics its entire complexity. We focus on cecal
ligation and puncture (CLP) because this has been consid-
ered to be the gold-standard model in sepsis research. We
summarize the results obtained using this model and their
contribution to our knowledge of the role of the innate
immune system in sepsis. We also propose different strate-
gies to bridge the gap between animal and human sepsis,
which will be useful for identifying promising new thera-
peutic targets for the treatment of sepsis.
The upsides and downsides of animal models of sepsis:
is CLP the gold standard?
Although sepsis is one of the most difficult clinical condi-
tions to model in animals, different animal models have
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TIMI-782; No. of Pages 11
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Local
Sterile causes SIRS
- Fever
- Tachycardia
Trigger
- Tachypnea
- Leucocytosis
Infection Sepsis
(proven or suspected)
Figure 1. General definitions of SIRS and sepsis. Local inflammation can be triggered by sterile causes or infections, but when the host response becomes uncontrolled
SIRS and sepsis can develop. SIRS evolves to severe SIRS and sepsis to severe sepsis when there is solitary or multiple organ failure. Finally, (septic) shock refers to a
condition in which there is refractory hypotension despite adequate fluid resuscitation. Recovery is possible at each stage of the disease, but the survival rate declines
substantially in the later stages of the disease.
been developed. These commonly follow one of three strat-
egies: (i) administration of exogenous toxins, such as zy-
mosan and lipopolysaccharadide (LPS); (ii) administration
of viable pathogens, such as bacteria and viruses; and (iii)
alteration of the endogenous protective barrier of the ani-
mal. The CLP model and the colon ascendens stent perito-
nitis (CASP) model are examples of models that disturb the
endogenous intestinal barrier. These approaches attempt
to reproduce the pathophysiological changes typically ob-
served in septic patients. However, not all animal models
mimic human sepsis as effectively (Table 1). For example,
early in the course of endotoxemia there is a strong and
rapid increase of several proinflammatory cytokines, in-
cluding tumor necrosis factor (TNF) and interleukins (ILs)
IL-1b and IL-6, in contrast to the lower and more prolonged
cytokine increase in sepsis patients. Thus, although these
models can give some insight into the process of sepsis,
they do not reproduce the full clinical complexity and
intrinsic heterogeneity of patients. Despite these limita-
tions, using animal models is the best available approach,
and it will remain essential for the development of new
therapies for sepsis.
Table 1 summarizes the advantages and drawbacks of
the most commonly used sepsis models. The CLP model is
one of the most stringent models of sepsis, and is consid-
ered by many investigators to be the crucial preclinical test
for any new treatment of human sepsis [7]. This model has
been used extensively over the past 30 years to study the
pathophysiology of sepsis [8]. Compared to other models,
CLP provides a better representation of the complexity of
human sepsis. CLP involves a combination of three insults:
tissue trauma due to laparotomy, necrosis caused by liga-
tion of the cecum, and infection due to the leakage of
peritoneal microbial flora into the peritoneum. This latter
results in peritonitis followed by translocation of enteric
bacteria into the bloodstream, which activates the inflam-
matory response, eventually leading to septic shock. One
advantage of CLP is that the pathogens originate from
within the host, therefore mimicking traumatic injury
leading to peritonitis in humans. The CLP technique
(Box 1) became popular because it satisfies many of the
essential criteria of a good sepsis model: a simple proce-
2
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Systemic
Severe
Shock
SIRS
Organ failure Hypotension
Severe Septic
sepsis shock
TRENDS in Microbiology
dure, polymicrobial nature with a localized infectious fo-
cus, and bacterial products released in the periphery that
lead to septicemia. Furthermore, CLP shows a high degree
of similarity to human sepsis progression [7]. Neverthe-
less, interlaboratory standardization is difficult due to
inadequate control of the bacterial challenge and other
variable factors, including age, sex and strain of the ani-
mals used and experimental variation (Box 1).
The other host barrier disruption model, CASP, also
causes acute polymicrobial septic peritonitis. A stent of
defined diameter is surgically inserted into the ascending
colon, and this causes continuous influx of enteric bacteria
into the peritoneal cavity. Similar to CLP, the severity of
the inflammatory response depends on several experimen-
tal parameters, for example the diameter of the stent [9].
However, the hemodynamic response to CASP-induced
sepsis is not as well characterized. Compared to CLP,
CASP is a relatively new model and fewer confounding
variables have been identified so far. Moreover, CASP is a
more difficult surgical procedure than CLP. In fact, lack of
handling uniformity is a weakness in reproducibility in the
CASP model.
Both septic peritonitis models generate an acute inflam-
matory reaction, but they have different pathophysiolo-
gies. Typically, induction of systemic cytokines is stronger
and bacterial counts are higher in CASP than in CLP [9].
The systemic cytokine profile in CLP resembles the pro-
tracted elevation of cytokine levels observed in sepsis
patients [10]. Differences in cytokine profiles between
CLP and CASP probably account for the different outcomes
observed in mice deficient for the receptor for type I inter-
ferons (IFNAR1 / mice) after induction of sepsis by CLP
or CASP [11,12]. It appears that the acute systemic hyper-
inflammatory response in CASP-induced sepsis [9] is more
similar to the strong immune response to endotoxemia,
whereas the inflammatory reactions observed in CLP re-
semble those detected in clinical sepsis.
The CLP model is important for sepsis research
Animal models are an indispensable tool for understand-
ing the molecular and genetic mechanisms of sepsis. The
availability of a variety of mutant mouse strains allows the
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Table 1. Advantages and disadvantages of the most commonly used experimental models for sepsis
Sepsis model Advantages
Endotoxemia model: Simple and reproducible
systemic administration
of LPS
Induced response is acute
Highly controlled and standardized model
Bacterial inoculum model Presence of a bacterium allows
insights into mechanisms of host
response to pathogens
CLP model Simple procedure
Presence of an infectious focus
Polymicrobial sepsis model
using the complete spectrum of
host enteric bacteria
Recreates human sepsis progression
with similar hemodynamic and metabolic
phases and the presence of both hyper- and
hypoinflammatory phases
Prolonged and lower elevation of cytokine
release, as in humans
Box 1. The CLP technique
The CLP technique was implemented by the group of I.H. Chaudry
more than 30 years ago [7,61] and has recently been described in
detail [62]. Cecum ligation is performed immediately below the
ileocecal valve, located at the junction between the large and small
intestines, which provides an inflammatory source of necrotic tissue
[63]. This is followed by a perforation of the cecum (Figure I). This
allows fecal material to leak into the normally sterile peritoneal cavity.
Subsequently, animals develop typical symptoms of sepsis and
usually die.
Despite clinical relevance of CLP and its widespread use in sepsis
research, it remains difficult to control the magnitude of the septic
challenge. The outcome after CLP is strongly dependent on several
factors during the procedure. These include (i) the percentage of
cecum that is ligated and thus the amount of necrosis that is induced;
(ii) the amount of the microbial dose that enters the peritoneum,
which depends on the number and size of punctures and the pressure
exerted on the cecal end; (iii) variability in surgical skills, for example
the size of the incisions made in the skin and abdominal muscle; (iv)
use of different anesthetic agents; (v) time of day (i.e. circadian
rhythm effects); (vi) sex, age and strain of the animals; (vii)
heterogeneity of the animal host response (i.e. the immune system
of the host can succeed in containing the infection within an abscess)
[7,9] and this prevents development of septic shock; and (viii)
supportive treatments such as fluid resuscitation and antibiotic
treatment. As a consequence, it is of great importance that the
procedure is performed with high consistency and reproducibility.
Unfortunately, details of CLP procedures are not routinely described
in published studies, although such information could explain and
avoid many contradictory results obtained by different laboratories.
Trends in Microbiology xxx xxxx, Vol. xxx, No. x
Disadvantages
LPS-mediated signaling is strictly TLR4-dependent
Does not reflect all complex physiological human responses
High, rapid and transient increase in cytokines, which differs
from human sepsis
Rodents are endotoxin resistant, whereas humans are very sensitive
Different hemodynamic response compared to human sepsis
Variability in dose, toxin, and route of administration
Growth and quantification of bacteria is needed before
administration
The single bacterium model does not reflect the diversity
and combinations of infectious agents that are present in
human sepsis
Humans are normally not challenged with a massive
bacterial load, but have a septic focus that intermittently
and persistently challenges the body with bacteria
High doses of bacteria induce an endotoxic instead of a
septic shock, due to the presence of LPS after rapid lysis
of the bacteria
Variability in bacterial load, route of administration and
bacterial strain
Abscess formation
Variability in severity due to differences in experimental
procedures (Box 1)
For example, the length of the ligated cecum is a major determinant
of mortality [64]. There is also a positive correlation between needle
size and mortality [61]. This is illustrated by two different studies
evaluating the role of IL-10 in sepsis using double-puncture CLP with
20-gauge and 22-gauge needles. Survival was significantly greater in
the 20-gauge needle group than in the 22-gauge group [45,65].
Therefore, it is essential that the CLP procedure applied is described
in great detail in publications. Nevertheless, the variability of the CLP
technique is also considered to be an advantage: the procedure can
be adapted to induce a range of severity, thereby permitting the
investigation of both acute and chronic sepsis.
(a) (b) (c) (d) (e)
TRENDS in Microbiology
Figure I. CLP procedure. (a) Animals are first anesthetized and the abdominal
area is shaved and disinfected. A midline laparotomy is then performed. (b) The
cecum is identified and exposed and a specified percentage of the distal end of
the cecum is ligated. The higher the percentage, the higher the mortality [64]. (c)
Next, the cecum is punctured. The more punctures and the bigger the needle
size, the higher the mortality [7,61]. The cecum is then gently compressed to
extrude a small amount of cecal content. Next, the cecum is returned to the
abdominal cavity. (d) The abdominal musculature is closed by applying simple
running sutures. (e) The abdominal skin incision is closed by applying metallic
clips to the skin. At this point, optional antibiotic therapy and fluid resuscitation
can be given.
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Table 2. Outcome of blocking innate immune components in CLP-induced sepsisa

Immune component Intervention Outcome b Refs
Cytokines
IL-6 KO mice x [66]
[67]
Anti-IL-6 Ab + [15]
x [68]
IL-10 KO mice [69]
Anti-IL-10 Ab [45]
+ [46]
IL-12 KO mice [44]
x [56]
Anti-IL-12 Ab [43]
IL-13 Anti-IL-13 Ab [70]
IL-17 IL-17 receptor KO mice [17]
Anti-IL-17 Ab + [16]
IL-27 EBI3 (subunit of IL-27) KO mice + [71]
Soluble IL-27 receptor fusion protein + [71]
IFN-g KO mice x [56]
KO mice plus antibiotics + [55]
Anti-IFN-g Ab +c [72,73]
x [55]
IFN-gR KO mice x [56]
TNF TNFR1 KO mice + [74]
TNFR2 KO mice [74]
TNFR1/2 KO mice + [75]
Anti-TNF Ab [41,42]
Anti-TNF Ab plus antibiotics x [76]
GM-SCF KO mice + [77]
MIF Anti-MIF Ab + [59]
HMGB1 d Anti-HMBG1 Ab + [78]
RAGE KO mice (major receptor for HMGB1) + [79–81]
Type I IFN IFNAR KO mice [12]
Chemokines (and receptors)
CCL2 Anti-CCL2 Ab – [13]
CCL5/CCR1 CCR1 KO mice + [82]
Anti-CCL5 Ab + [82]
CCR6 CCR6 KO mice + [83]
CCR8 CCR8 KO mice + [84]
CXCL1/CXCR1 CXCR1 KO mice [85]
CXCL2/CXCR2 CXCR2 KO mice + [86]
Anti-CXCL2 Ab + [87]
Proinflammatory enzymes
MMPs Broad-spectrum Ab + [88–90]
TIMP3 KO mice [91,92]
NOS1 KO mice [93]
NOS2 KO mice [94]
PTGS2 Anti-PTGS2 Ab x [95]
Toll-like receptors
TLR4 KO mice + [96]
TLR9 KO mice + [97]
a
Abbreviations: Ab, antibody; TNFR, tumor necrosis factor receptor; GM-CSF, granulocyte-macrophage colony stimulating factor; CCL, chemokine (C-C motif) ligand; CCR,
chemokine (C-C motif) receptor; chemokine (C-X-C motif) ligand; CXCR, chemokine (C-X-C motif) receptor; MMP, matrix metalloproteinase; KO, knockout; TIMP, tissue
inhibitor of MMP; NOS, nitric oxide synthase; PTGS, prostaglandin endoperoxidase synthase.
b
Symbols: x, no difference in CLP-induced mortality; +, improved survival after CLP-induced sepsis; –, decreased survival after CLP-induced sepsis.
c
Mortality was not tested in the study of Qiu et al. [73], but the authors concluded that inhibition of IFN-g activity in the CLP model is beneficial because it accelerated fibrin
deposition in cecal tissue, preventing bacterial spread and reducing the systemic inflammatory response.
d
Many agents have resulted in improved survival in CLP-treated animals, including neuropeptides [98], vagus nerve stimulation [99], green tea [100] and ethyl pyruvate
[101], probably all due to an indirect decrease in HMGB1 levels.

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study of the role of specific genes and has greatly contrib-
uted to our knowledge of the immune responses associated
with experimental sepsis. Here, we summarize the find-
ings obtained by targeting specific components of the im-
mune system in CLP-induced sepsis.
Hyperinflammatory phase of sepsis
Sepsis is caused by overactivation of the innate immune
system, and this leads to the release of large amounts of
inflammatory mediators including cytokines and chemo-
kines. This proinflammatory storm causes the release of
powerful secondary mediators, such as inflammatory
enzymes and reactive oxygen species, which further am-
plify the inflammatory process. Therefore, this initial im-
mune reaction is called the hyperinflammatory phase of
sepsis. The major proinflammatory cytokines, IL-6 and
TNF, and chemokines such as monocyte chemoattractant
protein (MCP) 1, increase following CLP [13]. Further-
more, high levels of IL-6, which also occurs in human
sepsis, correlate strongly with decreased survival after
CLP [14]. Blockade of IL-6 consistently increases survival
of mice following induction of sepsis by CLP [15]. Inhibition
of several other proinflammatory mediators has already
been tested, either by genetic or pharmacological means, to
determine their involvement in experimental sepsis in-
duced by CLP (Table 2).
The specific roles of various inflammatory proteins in
the outcome of polymicrobial sepsis induced by CLP are
listed in Table 2. Inhibiting some of these mediators
improves survival, which shows that they are negative
regulators of innate immune function in CLP-induced
septic peritonitis. Therefore, in vivo blockade of some of
these cytokines has been regarded as a potential therapeu-
tic strategy for the treatment of sepsis. However, the
results of research are often discordant (Table 2), possibly
due to differences in mouse strains and experimental
variables in the CLP procedure (Box 1). For example,
conflicting results were reported about the role of IL-17
in polymicrobial sepsis. One study claims that blockade of
IL-17 is protective in severe CLP-induced sepsis [16],
whereas a second study that used mice deficient in the
IL-17 receptor demonstrated a beneficial role for IL-17 in
CLP-induced non-severe sepsis [17]. This discrepancy
might be explained by the different degrees of severity
of sepsis that can be induced by CLP (Box 1). These two
studies could be reconciled by hypothesizing that IL-17 is
essential for host defense but detrimental in severe inflam-
matory conditions. Nevertheless, contradictory results
might also be due to the complexity of the disease. Redun-
dancy between mediators and the fact that several med-
iators can exert a beneficial or a detrimental effect,
depending on the inflammatory context, complicates data
interpretation.
Concomitant with the initial hyperinflammatory re-
sponse is a nearly simultaneous production of anti-inflam-
matory proteins, such as IL-10 and glucocorticoids (GCs),
which dampen and terminate the inflammatory response.
This is exemplified by a recent study in which mice defi-
cient for mitogen kinase phosphatase 1 (Mkp1), a GC-
induced anti-inflammatory protein, showed increased in-
flammation and mortality when subjected to CLP [18]. As a
Trends in Microbiology xxx xxxx, Vol. xxx, No. x
result of this anti-inflammatory response, most patients
survive the initial hyperinflammatory state and progress
quickly to a protracted period of immune suppression [19].
Hypoinflammatory phase of sepsis
Important factors contributing to the immunosuppressive
phase is the shutting down of signaling pathways in neu-
trophils and the rapid apoptosis of lymphocytes and den-
dritic cells (DCs) [19,20]. During this hypoinflammatory
phase of sepsis, patients are very susceptible to nosocomial
infections, and most die during this long stage of immuno-
suppression [21]. Therefore, prevention of sepsis-induced
apoptosis has become a target for therapy. CLP is an
appropriate model for investigating this because these
phenomena are also present in CLP mice [20,22]. Mice
that overexpress B-cell CLL/lymphoma (Bcl-2), an inhibi-
tor of apoptosis, are resistant to CLP-induced apoptosis
and show improved survival [23]. In addition, chemical
inhibitors of activated caspases also protect against apo-
ptosis in CLP-induced sepsis [19]. More recently, addition-
al strategies have been reported to reduce apoptosis-
induced depletion of immune cells. Pène and colleagues
showed that blocking the Toll-like receptors TLR2 and
TLR4 results in reduced apoptosis of splenic DCs after
CLP [24]. Furthermore, IL-15 therapy was shown to have
beneficial effects on the outcome of CLP-induced sepsis by
reducing apoptosis of immune cells and by increasing the
levels of interferon (IFN)-g [25]. Hence, IL-15 administra-
tion represents a potential novel therapy for sepsis. Also,
inhibition of the negative costimulatory molecule pro-
grammed-death 1 (PD-1), whose levels strongly increase
during sepsis, reverses immune dysfunction and improves
survival during CLP-induced sepsis [26]. These examples
illustrate that patients can benefit from anti-apoptotic or
immune-stimulatory therapies during the hypoinflamma-
tory phase of sepsis, instead of from therapies that reduce
the immune response [19,21].
Coagulation and complement systems
Sepsis affects not only the inflammatory pathways but also
the coagulation system and the complement pathways.
During sepsis, systemic inflammation is associated with
activation of procoagulant pathways; this favors the devel-
opment of microvascular thrombosis and disseminated
intravascular coagulopathy [2]. The levels of natural antic-
oagulants, such as activated protein C (aPC), antithrombin
and tissue factor pathway inhibitor, are decreased in most
patients with sepsis. This results in the development of a
procoagulant and antifibrinolytic state. Thus, clinical ap-
plication of agents that inhibit the coagulation pathway or
promote fibrinolysis could restore homeostasis and
improve outcome. Again, the CLP model has been helpful
in elucidating mechanisms targeting the coagulation sys-
tem during sepsis. For example, lavage with recombinant
tissue-type plasminogen activator (t-PA) enhanced intra-
abdominal fibrinolysis in a dose-dependent fashion in a
mouse CLP model [27]. Peritoneal lavage with aPC also
rebalanced coagulation and fibrinolysis and improved sur-
vival in CLP-induced polymicrobial sepsis [27].
Sepsis is further characterized by uncontrolled amplifi-
cation of all three complement pathways, as reflected by
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the appearance of complement activation products in the
circulation (C3a, C4a and C5a) [28]. These anaphylatoxins
have strong proinflammatory activities, especially C5a
[29]. It was reported that C1 inhibitor improved survival
in the CLP model [30]. The most important effect of inhi-
biting C1 is probably decreased generation of C5a. In
addition, blocking C5a or C5a receptor by administration
of a specific antibody significantly improved survival and
prevented the development of organ dysfunction after CLP
[31,32].
The CLP-induced polymicrobial sepsis model has con-
tributed much to our knowledge of the involvement of
immune components in sepsis disease, including the iden-
tification of new potential drug targets. However, care
must be taken when extrapolating results on the efficacy
of new therapeutic agents from animal models to humans.
This became clear when most of the promising therapeutic
approaches did not improve survival of sepsis patients [33].
Strategies to bridge the gap between bench and
bedside
Although the CLP model has greatly contributed to our
understanding of the pathophysiological and immunologi-
cal features of clinical sepsis, it does not reproduce the
whole spectrum of human sepsis. Many promising thera-
peutic agents that were effective in animal studies, includ-
ing high-dose GCs [34], LPS-targeting agents [35,36], and
selective blockers of inflammatory mediators, all failed to
demonstrate a similar benefit in human clinical trials [33];
very few therapies have been translated to the clinic.
Despite controversy, dotrecogin alfa (aPC, a coagulation
factor) was shown to decrease mortality in severe sepsis
[37]. In addition, treatment with low doses of GCs also
showed a favorable benefit-to-risk ratio in severe sepsis
cases [38].
The disagreement between animal experiments and
human studies can be attributed to misinterpretation of
preclinical data obtained from experimental studies and
the use of animal models that do not adequately mimic
human sepsis; importantly, the outcome in the clinical
setting is influenced by multiple factors, including genetic
background, therapeutic interventions, supportive care,
age, and pre-existing medical conditions [39]. Here we
propose different strategies that take into consideration
these premorbid factors so as to improve the CLP model as
a valid platform for identifying new therapeutic targets.
The need for a better understanding of the pathogenic
mechanisms driving sepsis
Most previous experimental therapies for sepsis have fo-
cused on attenuating the initial inflammatory response
and have ignored the progressive development of immuno-
suppression [40]. In fact, the inflammatory response has
both beneficial and deleterious effects. It causes organ
system dysfunction and ultimately mortality, but the in-
duction of inflammatory proteins is often indispensable to
battle the infection successfully. Therefore, an optimal
therapy should block the overactivation of the immune
system but not prevent clearance of the pathogen. This
might explain the increased mortality consequent to block-
ing proinflammatory cytokines in CLP-induced sepsis,
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such as in anti-TNF antibody pretreatment [41,42] or by
blocking IL-12 [43,44] (Table 2).
Nevertheless, activation of proinflammatory proteins is
counterbalanced by the production of endogenous anti-
inflammatory mediators that prevent excessive activation
of the inflammatory response. This is exemplified by neu-
tralization of endogenously produced IL-10 which resulted
in increased plasma levels of TNF and enhanced mortality
[45]. However, a delayed therapy with anti-IL-10 monoclo-
nal antibodies was successful after CLP-induced sepsis
[46]. This might be explained by the contribution of the
endogenous anti-inflammatory reaction to the initiation of
immunosuppression.
Indeed, an important factor contributing to the com-
plexity of sepsis and the difficulty of finding effective
therapies is the gradual switch from a pro- to an anti-
inflammatory state. Failure of immunosuppressive drugs
can be attributed to the absence of a hypercytokine re-
sponse at the time of drug treatment. Because most
patients die during the protracted immunosuppressive
phase of sepsis due to secondary nosocomial infections,
modulation of the immunosuppressive phase of sepsis
might aid in the development of new therapeutic strate-
gies. Understanding both phases of human sepsis requires
animal models in which the hyperinflammatory response
can be overcome, thereby permitting the host defense to be
studied during the more prolonged phases of sepsis. To this
end, CLP is the most appropriate model because it resem-
bles the progression from the hyper- to the hypoinflamma-
tory phase of human sepsis. Increased risk of developing
secondary infections during the protracted immunosup-
pressive stage also occurs in the CLP model [20,47]. Thus,
the CLP model has been adapted accordingly by several
groups to study the pathological effects of such infections
by challenging animals after CLP with the fungus Asper-
gillus fumigatus [48] or the Gram-positive bacterium Pseu-
domonas aeruginosa [47].
Several therapeutic strategies have been proposed to
prevent immunosuppression, of which neutralization of
apoptosis is probably the most appropriate. However, apo-
ptosis blockers have not been tested yet in patients. An-
other strategy to restore immune function is the
administration of immune stimulatory proteins during
the immunosuppressive phase, such as granulocyte colony
stimulating factor (G-CSF). A recent study of a small group
of sepsis patients showed a benefit of G-CSF therapy [49].
Many other potential therapies targeting immunosuppres-
sion in sepsis have been reported, some of which were
described above. Blocking the complement protein C5a
is also promising because it plays a key role in the devel-
opment of immune paralysis (Box 2).
The need for better simulation of the clinical situation in
humans
Many of the therapeutic failures can also be attributed to
initiating clinical trials on the basis of results obtained in
animal models that do not fully reflect the pathophysiology
of human sepsis. Rodents are housed in specific pathogen-
free facilities, are often inbred strains, and have the same
age and weight. Patients are far more heterogeneous with
respect to gender, age, immune and nutritional status, and
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Box 2. Neutralization of the complement component C5a as a new promising therapy in sepsis
C5a is a complement-derived anaphylatoxin. By exerting diverse
proinflammatory effects that affect chemotaxis and activation of
granulocytes and macrophages, and the release of antimicrobial
products from phagocytes, it protects the host against microorgan-
isms. However, excessive production of anaphylatoxins injures the
host and can even be life-threatening. This occurs early in sepsis
when the exaggerated release of C5a disturbs the immune functions
in neutrophils, but it can be prevented by anti-C5a treatment
immediately after CLP [31,32]. Increased generation of C5a also
contributes to other harmful outcomes of sepsis, including apoptosis
of thymocytes and adrenal medullary cells, and cardiomyopathy
(Figure I) [102].
In the experimental model of CLP, all three complement pathways
are activated [28]. Therefore, blockade of C5a has been considered to
be a potential therapy for sepsis and has been tested in the CLP
model. In vivo blockade of C5a greatly attenuates many of the
complications that develop following CLP. In rodents, neutralization
of C5a after CLP improves survival, protects against the loss of innate
immune functions of neutrophils, alleviates the cardiomyopathy
associated with sepsis, attenuates consumptive coagulopathy, and
reduces thymocyte apoptosis [22]. Diminished thymocyte apoptosis
reduces the progression to an immunosuppressive state, which is
characteristic of clinical sepsis and an important cause of death of
sepsis patients.

Sepsis

Complement activation

Classical pathway Lectin pathway Alternative pathway

(Immune complexes) (Bacterial polysaccharides) (Lipopolysaccharide)

C1 MBL, MASP1/2/3/, MAP19 C3b

C4, C2 C3 Convertase

C3
- Phagocytosis
- Viral neutralization
C3a C3b - Antibody response
- T cell activation

Anaphylatoxins C5 Convertase

C5a C5b + Cell lysis

- Uncontrolled inflammation
- Compromised neutrophil C6, C7,
functions C8, C9
- Thymocyte apoptosis
- Consumptive coagulopathy
- Apoptosis of adrenal
medullary cells
- Cardiomyopathy

TRENDS in Microbiology

Figure I. Sepsis-induced activation of the three complement pathways collectively results in the generation of the proinflammatory anaphylatoxins C3a and C5a. The
complement system can be activated by at least three different pathways, each of which leads to the cleavage of C3 and C5 by the C3 and C5 convertases. During sepsis,
however, excessive activation of the complement system can contribute to the detrimental symptoms observed in septic patients, including uncontrolled inflammation,
defective phagocyte functions, apoptosis of thymocytes and adrenal medullary cells, and consumptive coagulopathy.

supportive care than are commonly used laboratory mice.
These factors influence the clinical outcome in human
sepsis (Table 3). For example, most humans with sepsis
are more than 50 years old, whereas most mice used in
experimental sepsis models are typically aged 2–3 mo, the
equivalent of about 10 years of age in humans. Studies
using CLP have demonstrated that there is a relationship
between age and increased mortality [50]. In addition,
experimental animal models have provided a better un-
derstanding of the role of gender differences in the re-
sponse to CLP, indicating that female mice are more
resistant to CLP than are male mice [51]. Therefore, when
testing the efficacy of new drugs it is better to reproduce at
least some of this heterogeneity in animals.

7
TIMI-782; No. of Pages 11
Review
Table 3. Discrepancies between CLP and human sepsis that might account for failure of therapeutic sepsis strategies
Discrepancy CLP sepsis model
Age Usually young animals
Weight Equal
Gender Usually one gender
Immune status Optimal
Nutritional status Equal
Genetics Inbred
Social factors Seldom
Environment Defined, often pathogen-free
Onset Usually rapid
Causative insult Polymicrobial: mixture
of enteric bacteria
Antibiotics Sometimes
Treatment Usually early
Anticoagulant High
Comorbidities Seldom
Supportive care Seldom
Most importantly, unlike patients, experimental ani-
mals do not have comorbidities such as diabetes, renal
failure, or pre-existing immunosuppression. To develop
better preclinical sepsis models several groups have tried
to mimic some of the heterogeneity of humans by imple-
menting comorbid conditions in animals. Recently it has
been reported that untreated type 1 and 2 diabetes severe-
ly exacerbates mortality and sepsis-induced inflammation
following CLP [52,53]. In addition, Doi et al. showed that
pre-existing renal disease worsens outcome following CLP
[54].
Another potential component that could contribute to
the disparity in the development of novel effective thera-
pies is the lack of supportive therapeutic interventions in
experimental sepsis models. Humans are immediately
treated with different standard therapies including anti-
biotics, glucose control and correction of anemia. The
importance of these supportive treatments in the severity
of the sepsis response has been illustrated by using differ-
ent resuscitation strategies and by the use of antibiotics in
CLP. The use of antibiotics in the study of Romero et al. can
explain the increased resistance of IFN-g knockout mice to
CLP-induced mortality [55], in contrast to the study of
Echtenacher et al. in which no antibiotic treatment was
applied [56]. Furthermore, early and more aggressive and
intensive fluid resuscitation, which resembles the situa-
tion in patients, shows the most beneficial effects on sepsis
outcome induced by CLP [57]. Taken together, preclinical
models of sepsis must take into account the importance of
appropriate fluid resuscitation and antibiotic regimens.
A more intense resuscitation strategy also simulates
better the hemodynamic profile of human sepsis. Indeed,
sepsis is associated with hemodynamic abnormalities that
result in tissue hypoperfusion. Monitoring of these hemo-
dynamic changes is very important, although this is not
evident in animal models. Evaluating hemodynamic
parameters upon CLP have been performed using echo-
cardiography and implanted radiotelemeters [57]. By ex-
tended physiological monitoring in sepsis patients, several
stages in the severity of sepsis have been described, where-
8
Trends in Microbiology xxx xxxx, Vol. xxx, No. x
Human sepsis
Mostly neonates or elderly
Variable
Both, but women have a more active immune system than men men
Often suboptimal
Diverse
Heterogeneous
Often: smoking, drinking, stress
Variable
Usually slow
Gram-negative- and Gram-positive bacteria, viruses and fungi
Mostly used and often are ineffective
Usually late
Low tolerance
Common: hypertension,cancer, atherosclerosis, diabetes, pre-existing
immunosuppression
Common: antibiotics, fluid resuscitation, mechanical ventilation,
blood pressure support by vasopressors
as experimental CLP is more difficult to stage due to a
current lack of efficient monitoring. The outcome of CLP is
normally assessed through the mortality rate. In view of
this, and the large number of variables inherent to the CLP
procedure, high numbers of animals are required. It has
recently been predicted that biomarkers such as inflam-
matory cytokines might be helpful to allow mortality pre-
diction [4,58].
Moreover, new therapeutic agents should be studied in
infection models even after initiation of the septic process.
In most successful experiments the agent was given before
or shortly after the septic challenge, whereas patients are
usually diagnosed and treated after the onset of sepsis.
Nevertheless, some therapies, such as macrophage migra-
tion inhibitory factor (MIF) therapy [59], anti-HMGB1
(high mobility group box 1) antibodies [60] and anti-IL-
17A antibodies [16], showed beneficial effects even if treat-
ment was delayed several hours after the infection started.
Thus, it is clear that novel therapeutic treatments should
also be effective when administered after the onset of
sepsis. One should also take into account that sepsis
patients rapidly evolve into an immunosuppressive state,
again promoting the development of therapeutic anti-apo-
ptotic, immunostimulatory agents for the treatment of
sepsis. Several potential therapies that target the immu-
nosuppressive phase were successful in CLP. For example,
anti-PD-1 antibodies, administered 24 h post-CLP, pre-
vented apoptosis of lymphocytes and DCs and improved
survival [26].
To develop effective therapies for sepsis patients, inves-
tigations of the immunological and pathophysiological
mechanisms should be performed in models that resemble
human sepsis more closely than do current models (Table
3). The CLP model can be adapted in several ways, but
some limitations are inherent to CLP. Findings obtained
from the CLP model can be applied to patients in whom
sepsis is due to abdominal perforations, leading to perito-
nitis. However, other types of insults that activate other
pathways can also result in sepsis. For example, the fre-
quency of fungal sepsis has risen, which is alarming be-
TIMI-782; No. of Pages 11
Review
cause it has a poor prognosis [2]. Gram-positive pulmonary
infection models also deserve consideration because lung
infection is an important source of sepsis in humans. This
clearly indicates that sepsis is a group of distinct biochem-
ical disorders rather than a single physiologic syndrome.
Future research will be needed to identify different groups
of patients, possibly by the use of novel biomarkers, to
improve therapeutic treatment and clinical studies [3].
Concluding remarks and future perspectives
Sepsis is a major problem with high incidence and mortal-
ity rates. Appropriate animal models are crucial for study-
ing sepsis, although most such models are not directly
relevant for investigations of the pathophysiology of hu-
man sepsis. Nevertheless, the CLP model is one of the best
representations of human sepsis and has made important
contributions to our knowledge of the inflammatory com-
ponents involved in sepsis and to the identification of
therapeutic targets. The CLP model has been considered
to be the gold-standard model of sepsis. However, it does
not completely reproduce the complexity of human sepsis
and, despite the promising therapeutic strategies that
emerged from using this model, most therapeutic inter-
ventions have been ineffective. To improve the prospect of
developing effective treatments for sepsis we must first
gain a greater understanding of the pathogenesis of sepsis
– which is characterized by an early hyperinflammatory
state followed by a pronounced hypoinflammatory state.
Moreover, in view of the heterogeneity of human patients
we should incorporate the variables of age, weight, pre-
existing diseases, genetic background, and clinical sup-
portive care into the experimental CLP model.
Acknowledgments
The authors wish to acknowledge Dr. Amin Bredan for critical review of
the manuscript. Research in the authors’ laboratory is sponsored by the
Fund for Scientific Research-Flanders, the Interuniversity Attraction
Poles Program of the Belgian Science Policy (IAP VI/18), the Belgische
Vereniging tegen Kanker, and the VIB.
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