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Keywords:

  • clinical immunology;
  • dendritic cells;
  • innate immunity

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
  9. Supporting Information

A mouse model of polymicrobial sepsis induced by cecal content injection (CCI) was developed with the aim of gaining a better understanding of the mechanism of sepsis. This model has a similar survival pattern to the conventional model with the added benefits of ability to vary the severity of sepsis and greater consistency. Administration of 1-methyl-D-tryptophan (1-MT) to inhibit indoleamine 2,3-dioxygenase (IDO) in mice with CCI-induced sepsis increased the survival rate and tended to up-regulate IL-10/IL-12 serum concentrations. The effectiveness of 1-MT was confirmed by increases in IL-10 over IL-12 in bone marrow-derived dendritic cells (BMDCs) treated with LPS and 1-MT and a superior survival rate 24 hr after injection of these double treated BMDCs in the CCI-induced sepsis model. Therefore, CCI is both a useful and reliable technique for investigating polymicrobial sepsis. The present findings using this newly developed model suggest that inhibition of IDO alleviates the severity of polymicrobial sepsis and modulates the immune response even in cases of severe systemic septic inflammation.

List of Abbreviations
1-MT

1-methyl-d-tryptophan

CASP

colon ascendans stent peritonitis

BMDC

bone marrow-derived dendritic cell

CCI

cecal content injection

CLP

cecal ligation and puncture

DC

dendritic cell

FITC

fluorescein isothiocyanate

IDO

indoleamine 2,3-dioxygenase

IFN

interferon

LPS

lipopolysaccharide

mAb

monoclonal antibody

TNF

tumor necrosis factor

Sepsis is life-threatening and its pathophysiology provides insights into understanding hyper-reactive immune responses. There are several animal models that mimic the pathophysiological changes seen in septic patients. Since its development 30 years ago, CLP has been the most widely used model for experimental sepsis. Perforation of the cecum, an endogenous source of bacterial contamination, results in bacterial peritonitis, which is followed by bacteremia. CLP triggers septic shock and multi-organ failure in experiment animals [1, 2].

Despite its clinical relevance, CLP has several shortcomings that have so far precluded obtaining reliable and reproducible results. The severity of sepsis in CLP can vary according to the different methods deployed and the animals used (inter-experimental variations) [1]. In addition, the nature of the bacteria contaminating the peritoneum and surgical and anesthetic procedures can influence the survival rate in CLP-induced sepsis. These issues motivated us to develop a CCI model for polymicrobial sepsis.

Cecal content injection, being a modified CLP-induced type of sepsis model, displays a similar pathophysiology to CLP in regard to cytokine changes and survival rate. Thus its outcomes can be directly compared with previous CLP experiment-generated results. In our CCI model, 1-MT, an IDO inhibitor and modified DCs were tested with the aim of evaluating their effects on survival rates of septic mice. We have previously reported that IDO is a key enzyme that produces the pro-inflammatory cytokine IL-12 in DCs and that IDO activity in DCs has a pivotal role in the survival from LPS-induced septic shock [3-5].

Recently, certain immune cells, such as T-lymphocytes, dendritic cells and natural killer cells, have been tested as cell-based immunotherapy for cancers, rheumatism and allergies [6-9]. Among them, dendritic cells would be the most potent because they are the most specialized antigen-presenting cells for inducing T cell-mediated immune response by diverse antigens and they can serve as a link between innate and adaptive immune response [5, 10, 11]. In this paper, we present our findings concerning the effects of adoptive transfer of DCs and 1-MT in our newly developed CCI-induced polymicrobial sepsis model.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
  9. Supporting Information

Animals

Eight to ten week-old ICR male mice were purchased from Oriental Animals (Seoul, Korea), housed in an isolated pathogen-free environment and kept in 12 hr light and dark cycles for a week before their use. All experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee, Korea University.

Sepsis induced by cecal content injection

After killing the mice with CO2, cecectomy was performed and the cecum extracted just beneath the ileo-cecal valve through a midline abdominal incision using aseptic procedures. The cecal contents were extruded with a cotton swab into a Petri dish. After the cecal contents had been weighed, an appropriate volume of PBS was added to the dish to a final concentration of 20 or 30 mg/mL. The cecal contents were minced with the use of ground glass. Homogenized cecal contents (1 mL) were injected i.p. into awake mice (see Supplementary Video File). The cecal contents from one mouse could be used for more than 10 mice in the same batch. To verify the efficacy of cecal content injection in inducing polymicrobial sepsis, their survival rate and serum cytokine concentrations were compared with results from the CLP-induced sepsis model, currently the most widely adopted method [2]. After inducing sepsis, the mortality of the mice was monitored daily for 7 days.

Culture of bone marrow-derived dendritic cells

After the mice (8-week-old) had been killed with CO2, their bone marrow cells were flushed from their tibias and femurs and the red blood cells depleted with Red Blood Cell Lysing buffer (Sigma–Aldrich, St Louis, MO, USA). The cells were plated in 6-well culture plates at concentrations of 106 cells/well (well volume 2 mL) in RPMI 1640 supplemented with 10% heat-inactivated FBS, 100 U/mL streptomycin, 20 ng/mL recombinant murine granulocyte macrophage colony stimulating factor and 10 ng/mL recombinant murine IL-4 at 37°C in a 5% CO2 incubator. Floating cells were gently removed and fresh medium added on Days 3 and 5 of culture. On Day 6, non-adherent and loosely adherent cells were harvested by gentle trituration. On Day 7, 80% or more of the harvested cells expressed CD11c, a typical surface marker of BMDCs. LPS (200 ng/mL, Escherichia coli 055:B5, Sigma–Aldrich) was added to the cultured BMDCs for 24 hr. In cases where 1-MT (1 mg/mL, Sigma–Aldrich) was involved, 1-MT pretreatment was added 4 hr before the LPS. After washing the modified BMDCs (106 cells/mouse) with fresh media, they were injected i.p. immediately after injection of cecal contents into the mice.

Flow cytometry analysis

Harvested DCs were stimulated with LPS or LPS with 1-MT for 24 hr for flow cytometry analysis. The treated BMDCs were washed with PBS and resuspended in fluorescence-activated cell sorter washing buffer (2% FBS and 0.1% sodium azide in PBS). The resuspended cells were blocked with 10% (v/v) normal goat serum for 15 min at 4°C. For intracellular cytokine staining, the cells were treated with brefeldin A (10 µg/mL) for 4 hr, washed with 1% (v/v) FBS–PBS, stained with FITC- or PE-conjugated CD11c mAbs and then fixed in 4% (w/v) paraformaldehyde for 20 min at room temperature. Subsequently, the cells were washed twice in a staining buffer and then rendered permeable in 100 µL of 0.1% saponin in 1% FBS–PBS. They were then incubated with FITC- (or PE-) conjugated anti-IL-12 p40/p70 and anti-IL-10 mAbs for 30 min at 4°C. The stained cells (105 cells) were analyzed using the FACSCalibur system (BD Biosciences, Franklin Lakes, NJ, USA). To calculate the percentage of positive cells, a proportion of 1% false-positive events was accepted.

Statistical analysis

The data was expressed as the mean ± SEM in bar graphs and as minimum, maximum, median, 25% and 75% in box-and-whiskers graphs. ANOVA and Student t-test were used to compare the experimental and control groups. Comparisons between the groups were performed using a Tukey's multiple comparison test. For analysis of the survival rate, a Kaplan–Meier estimator was used. Statistical significance was determined as P-value less than 0.05.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
  9. Supporting Information

To compare the effect of the two methods, cecal ligation and puncture (CLP, n = 90) and cecal content injection (CCI, n =130), on polymicrobial sepsis, we analyzed the time course and variation in survival rate of the experimental group. The time courses of the survival rates were similar for both methods. Mortality was highest during first the two days and became stable thereafter. No mice died between 4 and 7 days after CCI or CLP. Two mice that had received sham CLP (n = 40) died in the first 2 days from unidentified causes; however, no mice that had received only vehicle injection (controls for CCI) (n = 40) died. We induced mid-grade sepsis by the CLP method and the survival rate was similar to a previous report [2]. Because the severity of CLP-induced sepsis can be varied by changing the level of cecum ligation, we varied the severity of CCI-induced sepsis by changing the dose of cecal contents (Fig. 1a). As shown in Figure 1b, the inter-group survival variation was less for CCI than for CLP. For example, the standard deviations of survival rate in CCI- and CLP-induced sepsis were 5.8% (seven groups) and 15.4% (six groups), respectively, at 72 hr. In the mice with CCI-induced sepsis, serum concentrations of TNF-α and IFN-γ increased and reached their peaks (745.0 ± 193.6 and 65.4 ± 10.0 pg/mL) at 18 and 12 hr, respectively (Fig. 1c). In comparison, in the endotoxin shock model, serum concentrations (pg/mL) of TNF-α were 12 ± 2.1 before LPS injection, 795.5 ± 165.5 at 1 hr, 790.5 ± 323.6 at 2 hr, and 101.6 ± 10.5 at 5 hr after LPS injection (50 mg/kg, each n = 4).

image

Figure 1. Comparison between CLP- and CCI-induced polymicrobial sepsis in mice. (a) The time courses of the diseases were similar in both CLP (n = 90) and CCI (n = 130) models. The fatality rate was high in the first two days and stabilized thereafter. No mice died after vehicle injection alone (n = 40) in the CCI model control group, but two died after sham surgery (n = 40) in the CLP model control group. (b) Inter-group variation in survival rates was smaller in the CCI (30 mg/mL) model than in the CLP model. (c) Serum concentrations of TNFα and IFN-γ increased, peaking 18 and 12 hr, respectively, after initiation of CCI-induced sepsis.

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In the CCI model, 1-MT pretreatment (24 hr before inducing sepsis) significantly increased the survival rate of CCI-induced sepsis (n = 40, Fig. 2). In mice with polymicrobial sepsis induced by CLP, both IL-12 and IL-10 increased, peaking at 12 hr after CLP, then stabilizing until 48 hr (unpublished data). In order to evaluate the effect of 1-MT pretreatment on CCI-induced polymicrobial sepsis, IL-12 and IL-10 serum concentrations were measured at 48 hr after CCI; concentrations of both were found to be significantly increased. 1-MT pretreatment boosted the serum concentration of IL-10 more than that of IL-12, and the IL-10/IL-12 ratio amplified this tendency. The serum concentration of IFN-γ at 18 hr after CCI was increased, but was mildy reversed by 1-MT pretreatment (Fig. 3); this was not a statistically significant finding.

image

Figure 2. Effect of 1-MT on the survival rate in CCI-induced sepsis. Pretreatment with 1-MT (n = 40) significantly increased the survival rate in CCI-induced sepsis. (*, P < 0.05).

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image

Figure 3. Changes in serum cytokines in the CCI-induced sepsis models. (a and b) At 48 hr, both IL-12 and IL-10 serum concentrations were significantly increased in the CCI-induced sepsis mice. (c) 1-MT pretreatment increased the IL-10/IL-12 cytokine ratio in comparison to the ratio in the septic mice. (d) At 18 hr, with 1-MT pretreatment serum concentrations of IFN-γ, an important cytokine in Th1 immune responses, had decreased. (*, P < 0.05; **, P < 0.01; each group n > 8).

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We previously reported that IDO in DCs has a pivotal role in the pathogenesis of LPS-induced septic shock. Here, the effect of BMDCs modified by LPS with/without 1-MT on CCI-induced sepsis was tested. Cultured BMDCs were modified by LPS for 24 hr with/without 1-MT pretreatment. The 1-MT treated BMDCs appeared to induce a greater increase in expression of IL-10 than of IL-12 (Fig. 4a). After washing the modified BMDCs (106 cells/mouse) with fresh media, we injected them i.p. into mice with CCI-induced sepsis. Adoptive transfer of BMDCs treated with LPS alone reduced the survival rate of these mice at 24 hr. However, BMDCs treated with LPS and 1-MT pretreatment increased the survival rate. The difference in survival rates at 24 hr between the two groups was significant (Fig. 4b and c).

image

Figure 4. Effect of i.p. injection of BMDCs on the survival of mice with CCI-induced sepsis. (a) Cultured BMDCs stimulated by LPS expressed IL-12 and IL-10. 1-MT pretreatment of stimulated BMDCs slightly increased IL-10, but reduced IL-12. (b and c) I.p. injections (106 cells/mouse) of BMDCs stimulated by LPS alone lowered the survival rate in mice with CCI-induced sepsis. However, injections of stimulated BMDCs pretreated with 1-MT significantly boosted the survival rate after 24 hr in mice with CCI-induced sepsis (*, P < 0.05; each group n = 20).

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
  9. Supporting Information

Several experimental animal models have been used to mimic the pathophysiological changes seen in septic patients and thus study the underlying mechanisms of sepsis. Among them, the CLP model replicates the nature and clinical course of septic patients after trauma and perforation of their gastrointestinal tracts. Therefore, CLP has been considered as a gold-standard model for inducing sepsis for research purposes [1, 12]. However, as several review papers have previously pointed out, CLP has certain shortcomings, such as varying severity of sepsis and abscess formation, which leads to unreliable results. The severity of sepsis varies according to the length of cecum ligated, the number of punctures, individual surgical skills, and different anesthetic agents. In addition, abscess formation occurs randomly, resulting in local rather than systemic CLP-induced inflammation [1, 12-14].

For inducing polymicrobial sepsis, CASP and the fecal pellet model have been less popular methods than CLP. CASP induces diffuse peritonitis and therefore has a lower incidence of local abscess formation in the peritoneum [15]. However, CASP also entails variability in surgical skills and anesthetic agents. The fecal pellet model has a tendency to induce peritoneal abscess formation rather than sepsis [16]. Thus, the LPS injection is the simplest model for the study of sepsis and ha good reproducibility. However, LPS-induced sepsis differs from that induced by the CLP model and of septic patients in several significant areas, such as the profiles of cytokine release [14, 17].

We have modified the gold standard CLP model, but retained an endogenous source of infection (cecal contamination). Induction of sepsis by CCI eliminates most variations that arise from the surgical skills of different experimenters and anesthetic agents in the CLP and CASP models. In addition, it induces diffuse peritonitis and does not cause local abscess formation in the peritoneum. Like LPS-induced sepsis, CCI is a simple method that allows the experimenter to control the severity of disease by changing the amount of cecal contents injected. Like CLP, CCI induces sepsis by contamination with an endogenous microbial source. In our serum cytokine analysis, the time course of the increments in serum TNF-α in CCI was similar to that found in a CLP experiment, the results of which were published previously [18]. One difference between CCI and CLP is the effect of necrosis of the ligated cecum. However, the necrotic tissue in the latter model would have a minimal effect on survival from the experimental sepsis given that mice tolerate cecal ligation without puncture quite well [12]. We believe that the CCI method for inducing polymicrobial sepsis is more reliable and reproducible than CLP, CASP, or the fecal pellet injection method.

Using the CCI model to induce polymicrobial sepsis, we tested the effect of 1-MT on survival rates. The compound 1-MT is a competitive antagonist of IDO, a rate-limiting enzyme of tryptophan metabolism. IDO is expressed in antigen-presenting cells, such as macrophage and dendritic cells, and is closely involved in their maturation and priming of T cells [4]. Recently, it has been shown that IDO is also expressed in endothelial cells [19]. IDO activity is reportedly increased in septic patients, and correlates with the severity of sepsis [20]. We previously proposed that inhibition of IDO reduces the mortality of endotoxin-induced septic shock in mice [5]. In this study, 1-MT pretreatment reduced the mortality rate of polymicrobial sepsis. However, the mechanism of 1-MT's effect on sepsis is still hotly debated [4, 5, 19, 21]. IDO induced by TLR-4 stimulation produces a pro-inflammatory cytokine (IL-12). However, blockade or knock-out of IDO increases anti-inflammatory cytokine concentrations (IL-10), and accordingly increases the IL-10/IL-12 ratio in mice [5]. Another possible mechanism would be that the kynurenine produced by IDO in endothelial cells induces vasodilatation, and to some extent, contributes to septic shock along with nitric oxide [19].

The profile of serum IL-12 and IL-10 in CCI-induced sepsis is not as clear as it is in LPS-induced septic shock. This might be a consequence of differences between endotoxin-induced and polymicrobial sepsis or between inbred C57BL/6 and outbred ICR mice [22]. Here, we used outbred mice in order to reduce the immunologically skewed effect of inbred mice, a factor which has been pointed out as a significant possible explanation for the differences in findings between animal studies and clinical patients [12, 14]. Nevertheless, 1-MT pretreatment significantly increased the survival rate in polymicrobial sepsis. Further study is required to shed light on the precise mechanism.

We conducted additional experiments to clarify whether immunomodulation of 1-MT in DCs affects survival in polymicrobial sepsis. We stimulated cultured DCs by LPS on TLR-4 signaling, with/without 1-MT pretreatment. DCs in both mice and humans not only increases production of Th1 cytokines induced by LPS stimulation, but also boosts production of Th2 cytokines induced by LPS stimulation with 1-MT pretreatment [5, 21, 23]. In this study, adoptive transfer of DCs treated with LPS and 1-MT in cultured DCs significantly increased the survival rate in CCI-induced sepsis. Although the effect of 1-MT might result from both IDO-dependent and IDO-independent mechanisms [5, 21], we suggest that adoptive transfer of in vitro modified DCs can soothe and alleviate the normal immunological reaction and resultant clinical symptoms by competing with endogenous DCs and that adoptive DCs may thus alleviate disease severity. DCs may be a useful pharmacological target for manipulating the immune response in polymicrobial sepsis.

In conclusion, CCI is both a useful and reliable method that could prove to be a worthy successor of the CLP model for the study of polymicrobial sepsis. Using the CCI model, we concluded that immunomodulation by 1-MT increases the survival rate in both polymicrobial sepsis and endotoxin septic shock and that injection of cultured DCs modulates the immune response in vivo even in cases of severe systemic sepsis.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
  9. Supporting Information

This research was supported by Core Research Support for Senior Researchers from the National Research Foundation of Korea (2010-002-7564), funded by the Ministry of Education, Science and Technology and a Korea University grant.

REFERENCES

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  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
  9. Supporting Information

Additional supporting information may be found in the online version of this article at the publisher's web-site:

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mim12081-sm-0001-SuppMovie-S1.mov33754KMovie S1. Technique of cecal content injection.

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