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. 2014 Nov;82(11):4487–4495. doi: 10.1128/IAI.02113-14

Blockade of Indoleamine 2,3-Dioxygenase Reduces Mortality from Peritonitis and Sepsis in Mice by Regulating Functions of CD11b+ Peritoneal Cells

Masato Hoshi a,c,✉, Yosuke Osawa b, Hiroyasu Ito c, Hirofumi Ohtaki c, Tatsuya Ando c, Manabu Takamatsu d, Akira Hara d, Kuniaki Saito e, Mitsuru Seishima c
Editor: B A McCormick
PMCID: PMC4249307  PMID: 25114116

Abstract

Indoleamine 2,3-dioxygenase-1 (Ido), which catalyzes the first and limiting step of tryptophan catabolism, has been implicated in immune tolerance. However, the roles of Ido in systemic bacterial infection are complicated and remain controversial. To explore this issue, we examined the roles of Ido in bacterial peritonitis and sepsis after cecal ligation and puncture (CLP) in mice by using the Ido inhibitor 1-methyl-d,l-tryptophan (1-MT), by comparing Ido+/+ and Ido−/− mice, or by using chimeric mice in which Ido in the bone marrow-derived cells was deficient. Ido expression in the peritoneal CD11b+ cells and its metabolite l-kynurenine in the serum were increased after CLP. 1-MT treatment or Ido deficiency, especially in bone marrow-derived cells, reduced mortality after CLP. Compared to Ido+/+ mice, Ido−/− mice showed increased recruitment of neutrophils and mononuclear cells into the peritoneal cavity and a decreased bacterial count in the blood accompanied by increased CXCL-2 and CXCL-1 mRNA in the peritoneal cells. Ido has an inhibitory effect on LPS-induced CXCL-2 and CXCL-1 production in cultured peritoneal cells. These findings indicate that inhibition of Ido reduces mortality from peritonitis and sepsis after CLP via recruitment of neutrophils and mononuclear cells by chemokine production in peritoneal CD11b+ cells. Thus, blockade of Ido plays a beneficial role in host protection during bacterial peritonitis and sepsis.

INTRODUCTION

Sepsis is a systemic inflammatory response syndrome induced by microbial infection (1, 2). The pathogenesis of sepsis involves a progressive and dynamic expansion of a systemic inflammatory response to bacterial infection (3). Neutrophils are major leukocytes that are promptly recruited to the inflamed site in response to infection or tissue injury. These cells are ideally suited to the elimination of pathogenic bacteria owing to their capability for phagocytosis and releasing the stores of granular lytic enzymes and antimicrobial polypeptides into the phagolysosome (4). In this way, migrating neutrophils may control bacterial growth and, consequently, prevent bacterial dissemination and death of the host (5). However, once the host fails to restrict the pathogens to a localized area, the pathogens and/or their products may spread systemically and result in the death of their host (6). Previous reports have demonstrated that the severity of sepsis induced by cecal ligation and puncture (CLP) (7) or by Staphylococcus aureus inoculation (8) is closely associated with reduced neutrophil migration to the infection focus. Therefore, to inhibit bacterial growth in the local site, neutrophils must migrate to the site of infection in response to chemotactic factors, such as chemokine (C-X-C motif) ligand 2 (CXCL-2) and chemokine (C-X-C motif) ligand 1 (CXCL-1). These chemokines, which are secreted from macrophages, neutrophils, and epithelial cells in response to endotoxin and various proinflammatory cytokines (9, 10), have been identified as chemoattractants of neutrophils in vitro and in vivo (11, 12) and have pathophysiological roles in several inflammatory disease states, including endotoxemia-induced lung injury (13), glomerulonephritis (14), and bacterial meningitis (15).

Indoleamine 2,3-dioxygenase-1 (Ido), which catalyzes the first and limiting step of tryptophan (Trp) catabolism, is induced in various cell types during infection, especially in response to gamma interferon (IFN-γ) signaling and/or bacterial components, such as Toll-like receptor (TLR) ligands, and plays a pivotal role in immune tolerance (16). Ido has been thought to have beneficial effects. For example, recent studies have shown that the TLR3 ligand poly(I·C) induces Ido activation in astrocytes, resulting in an antiviral response (17). IFN-γ-induced Ido also has an antiviral effect in measles virus infection of epithelial and endothelial cells (18). However, disadvantageous functions of Ido have also been reported. Ido is expressed in human (19) and mouse (20) tumor cells, dendritic cells (DCs) (16), and macrophages following microbial (21) or viral (22) infections, and Ido inhibition improves the pathophysiology of those ailments. Favorable effects of Ido inhibition are also reported in human immunodeficiency virus (HIV)-infected patients (23) and in major-trauma patients (24). Ido activation by CTLA-4 stimulation from the regulatory T cells or DCs is involved in viral increase in tissues from simian immunodeficiency virus (SIVmac251)-infected macaques (25). The Ido inhibitor 1-methyl-d,l-tryptophan (1-MT) may enhance anti-HIV immunity (26). Ido blockade protects mice against lipopolysaccharide (LPS)-induced endotoxin shock, in association with modulation of interleukin 12 (IL-12) and IL-10 production in DCs (27). In addition, several other studies suggest that Ido-expressing cells deplete Trp from the extracellular milieu and secrete Trp metabolites (including l-kynurenine [l-Kyn], 3-hydroxy-kynurenine, 3-hydroxyanthranilic acid, and quinolonic acid), which induce T cell apoptosis and suppress immune responses in vitro (28–30). Therefore, whether induction of Ido always has beneficial effects for the host and how Ido induces immune tolerance are not clear.

The CLP model is the most widely used rodent model for experimental peritonitis and sepsis. The model is considered to be realistic for the induction of polymicrobial sepsis in experimental settings to study the underlying mechanisms of sepsis (31). In this study, the roles of Ido in immune regulation were examined in polymicrobial sepsis induced by CLP by using Ido−/− mice or the Ido inhibitor 1-MT. We demonstrated that the induction of CXCL-1 and CXCL-2 in peritoneal cells by CLP was increased by Ido inhibition, resulting in increased recruitment of neutrophils and mononuclear cells to the local infectious focus, which suppresses progression to sepsis.

MATERIALS AND METHODS

Mice.

Eight- to 10-week-old male mice were used in this study. Ido1 gene-deficient (Ido−/−) mice on a C57BL/6J background were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Mice that were homozygous null (Ido−/−) by targeted disruption of the Ido gene were selected from the offspring of heterozygous-homozygous matings by genotyping by performing PCR of tail DNA. C57BL/6J mice obtained from Japan SLC (Shizuoka, Japan) were used as wild-type (Ido+/+) controls. For blockade of Ido, mice were administered 1-MT (5 mg/ml) in their drinking water starting from 3 days before or from just after CLP, and on average, each mouse consumed 3.5 ml/day. l-Kyn was administered as described previously (19, 32). Briefly, mice were intraperitoneally injected with l-Kyn (20 mg/kg of body weight; Sigma-Aldrich) 24 h before CLP. All animal experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of Gifu University (approval number 26-8).

Mouse sepsis model.

Sepsis was induced by CLP as previously reported (33). Briefly, mice were anesthetized by intraperitoneal administration of pentobarbital diluted in PBS (1.25 mg/ml; 200 μl), and a midline incision (1 cm) was made on the anterior abdomen. The cecum was exposed and ligated below the ileocecal junction without causing bowel obstruction. A single puncture was made using a 22-gauge needle to induce septic injury. Pressure was applied (the cecum was squeezed) to allow the cecum contents to be expressed through the puncture. The cecum was placed back in the abdominal cavity, and the peritoneal wall and skin incision were closed. The sham-operated animals underwent identical laparotomy without cecum ligation or puncture. Mortality after CLP was monitored in Ido−/− mice (n = 25), Ido+/+ mice (n = 23), 1-MT-treated mice (n = 17), and vehicle-treated mice (n = 15). To obtain samples, the animals were anesthetized and humanely killed at the indicated times. In another experimental animal model, sepsis was induced by inoculation of S. aureus (ATCC 25923). Briefly, mice were intravenously injected with S. aureus (1 × 108 CFU in 100 μl phosphate-buffered saline [PBS]). Mortality after the infection was monitored in Ido−/− mice (n = 12), Ido+/+ mice (n = 12), 1-MT-treated mice (n = 12), and vehicle-treated mice (n = 12).

Bacterial counts in the peritoneal exudate and in blood.

The peritoneal exudate (which was harvested by introducing 1.5 ml of PBS-EDTA [1 mM]), blood, and feces were collected under sterile conditions, and aliquots of serial dilutions of these samples were planted on brain heart infusion agar plates (Biovalley, Marne la Vallée, France). The plates were incubated at 37°C in a 5% CO2 atmosphere, and CFU were counted after 18 h. The results are expressed as the median log CFU per ml of the peritoneal exudate or blood and as the median log CFU per mg of feces. The samples were collected from sham-operated (n = 6) or CLP-operated (n = 7) Ido−/− or Ido+/+ mice.

Bacterial killing by neutrophils.

The bactericidal capacity of neutrophils was assessed as previously reported (34). Briefly, Ido+/+ and Ido−/− mice were intraperitoneally injected with thioglycolate (4%) to obtain peritoneal neutrophils. The induced neutrophils were harvested 2 h later by washing the peritoneal cavities with PBS. Cell viability was >98%, and the cell population consisted of neutrophils, representing >85% of the total leukocytes.

The bacterial suspension (Escherichia coli ATCC 25922) was added to the isolated Ido+/+ and Ido−/− neutrophil culture (2 × 106 bacteria and 1 × 106 neutrophils in 1 ml PBS) in 1.5-ml tubes, and the mixture was incubated for 3 h at 37°C with mild shaking. As a control, the bacterial suspension was incubated under the same experimental conditions in the absence of neutrophils. Bacterial viability was assessed by serial log dilutions and plating on brain heart infusion agar plates. CFU were counted after 18 h, and the results are expressed as the number of viable bacteria.

Leukogram.

Blood diluted in EDTA and peritoneal exudate were collected 6 h after CLP. Total counts were performed using a Neubauer chamber. Differential cell counts were conducted on slides stained with May-Gruenwald-Giemsa (Sysmex, Kobe, Japan). Differential counts were performed under oil immersion microscopy, where 200 cells were counted for the determination of the percentage of neutrophils and mononuclear cells present in blood and peritoneal exudate. The results are expressed as means and standard deviations (SD) of cells counted per milliliter.

Measurements of l-Kyn.

l-Kyn was measured using high-performance liquid chromatography (HPLC) with a spectrophotometric detector (UV-8000; Tosoh, Tokyo, Japan) as described previously (19, 32).

Cell preparation and culture.

Cells from the peritoneal cavity were harvested by introducing 1.5 ml of PBS-EDTA (1 mM). The isolated peritoneal cells were plated on 96-well culture plates (1 × 105 cells/well) or sterile slides in RPMI 1640 medium (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Boehringer Mannheim Biochemica, Manheim, Germany), 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) at 37°C in 5% CO2 for 2 h and washed with PBS. In some experiments, fractionation of the isolated peritoneal cells was performed using MACS MagneticBead columms (Miltenyi Biotec, Bergisch Gladbach, Germany) with antibodies against CD11b, CD11c, Gr-1, and DX5, according to the manufacturer's instructions. For LPS treatment, the cells were then treated with 0.1 μg/ml LPS from E. coli O55:B5 (Sigma-Aldrich, St. Louis, MO, USA) for 6 h.

Quantitative real-time reverse transcription (RT)-PCR.

Total RNA was isolated from peritoneal cells from sham- or CLP-operated mice, from cultured peritoneal cells treated or not with LPS for 6 h, or from fractionated cells using the RNeasy minikit (Qiagen, Hilden, Germany).

The isolated RNA was transcribed into cDNA with a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). The resulting cDNA was used as a template for real-time RT-PCR, along with primer-probe sets for Ido, CXCL-2, CXCL-1, MCP-1, CXCL-5, and IL-17 (TaqMan Gene Expression Assays; Applied Biosystems) and 2× TaqMan Universal PCR master mix (Applied Biosystems) according to the manufacturers' recommendations. The expression levels of the respective genes were normalized to those of 18S rRNA (Applied Biosystems) as an internal control in the reaction. All reactions were performed in duplicate. The sample value was expressed as the median interquartile range (IQR) and shown as a scatter plot.

Determination of chemokine levels.

CXCL-2 and CXCL-1 levels were detected in the sera of mice that were operated on or in the culture media for peritoneal cells by enzyme-linked immunosorbent assay (ELISA) according to the recommendations of the manufacturer (R&D Systems, Minneapolis, MN, USA).

Immunofluorescence assay.

Cells on the slides were fixed in 4% paraformaldehyde for 20 min. After incubation with 0.1% Triton X-100 for 30 min, the cells were blocked using 1% bovine serum albumin (BSA) and 0.1% Tween 20 containing PBS. Subsequently, the cells were incubated with primary antibodies against Ido and CD11b (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at 4°C. The anti-Ido polyclonal antibody was generated by the peptide H-CMKPSKKKPTDGDKS-OH in rabbits as described previously (19, 32). After washing with 0.1% Tween 20 containing PBS, the cells were incubated with the secondary antibodies fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG and rhodamine-conjugated anti-rabbit IgG (DakoCytomation, Carpinteria, CA, USA). 4,6-Diamino-2-phenylindole (Dojindo, Tokyo, Japan) was used for nuclear staining. The fluorescence was visualized using a BX51 fluorescence microscope equipped with a DP70 digital camera (Olympus, Tokyo, Japan). To determine the number of CD11b+ cells in the peritoneal cavity, the percentage of CD11b+ cells present in the peritoneal exudate was determined by counting 200 cells, and the total number of cells multiplied by the percentage of CD11b+ cells was calculated as the total number of CD11b+ cells.

BMT.

Bone marrow (BM) transplantation (BMT) was performed on 5-week-old male mice as described previously (32). The recipient mice (Ido+/+ and Ido−/− mice) were irradiated in fractionated doses (5 Gy twice with a 4-hour interval) and reconstituted with whole BM cells by injection (5 × 106 BM cells from Ido+/+ or Ido−/− donor mice) via the tail vein. These BMT mice were maintained under specific-pathogen-free conditions and given 500 U/ml of gentamicin sulfate (Invitrogen, Carlsbad, CA, USA) and 100 μg/ml of polymyxin B sulfate (Kayaku Co., Tokyo, Japan) in drinking water for 4 weeks after BMT. The animals were subjected to CLP, and mortality was recorded (at least 20 mice died in each group).

Statistical analyses.

The survival rates of mice were analyzed by the Kaplan-Meier method. Statistically significant differences between 2 groups were determined using Student's t test, and those among more than 3 groups were determined using one-way analysis of variance (ANOVA). Bacterial counts were reported as the median log CFU and were analyzed using the Mann-Whitney U test. StatView 4.5 software was used for these statistical analyses. The criterion for statistical significance was a P value of <0.05.

RESULTS

Ido inhibition suppressed CLP-induced mortality and reduced bacterial levels in the blood.

We first examined whether inhibition of Ido has beneficial effects on reducing the impact of sepsis. Treatment with 1-MT, an inhibitor of Ido, given either before (Fig. 1A, top) or after (Fig. 1A, bottom) CLP improved the survival rate following surgery, as did the knockout of Ido (Fig. 1B, top). Administration of l-Kyn reversed the Ido−/− phenotype (Fig. 1B, bottom). Similarly, the inhibition or knockout of Ido also improved the survival rate of S. aureus-induced sepsis (Fig. 1C). Serum l-Kyn levels significantly increased after CLP in Ido+/+ mice, and knockout or inhibition of Ido reduced the induction of l-Kyn (Fig. 2). These results indicate that l-Kyn production following Ido activation by CLP is involved in mortality. The bacterial load in the peritoneal exudate from Ido−/− mice 6 or 24 h after CLP was comparable to that from Ido+/+ mice (Fig. 3, bottom). In contrast, Ido−/− mice showed a marked decrease in the amount of bacteria in the blood compared with Ido+/+ mice (Fig. 3, top). Furthermore, there was no difference in the bacterial load in feces between Ido+/+ mice and Ido−/− mice (data not shown). In addition, Trp supplementation did not affect the intestinal bacterial growth on the minimal-medium plates (data not shown). These results suggest that CLP induces peritonitis and sepsis in Ido+/+ mice, as previously reported (35–37), and that the bacterial infection is regulated in the local infection focus in Ido−/− mice, resulting in the improved survival rate.

FIG 1.

FIG 1

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Inhibition or deficiency of Ido suppresses mortality after sepsis. (A) Wild-type mice underwent CLP. 1-MT or vehicle was administered 3 days before (top) or a few hours after (bottom) surgery. (B) Ido+/+ or Ido−/− mice underwent CLP (top). l-Kyn was administered 24 h before CLP in Ido−/− mice (bottom). S. aureus was inoculated into Ido+/+ or Ido−/− mice treated or not with 1-MT. The survival rates of the animals are shown. Statistically significant differences between the groups were determined using the log-rank test.

FIG 2.

FIG 2

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Inhibition or absence of Ido decreased the induction of serum l-Kyn levels after CLP. 1-MT-treated (24 h before CLP) and vehicle-treated (Sham) mice (A) or Ido+/+ or Ido−/− mice (B) underwent CLP. The animals were humanely killed at the indicated times. Serum l-Kyn levels were measured by using HPLC. The data are means and SD from at least 5 independent experiments. *, P < 0.01 using ANOVA.

FIG 3.

FIG 3

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Ido is involved in the bacterial load after CLP. Ido+/+ and Ido−/− mice underwent CLP. The numbers of bacteria in the blood (top) and in peritoneal exudate (bottom) were determined 6 or 24 h after CLP. The results are expressed as the median log CFU/ml in the peritoneal exudate or blood. *, P < 0.05 compared with Ido+/+ CLP mice, using a Mann-Whitney U test. ns, not significant.

To elucidate the mechanisms by which Ido contributes to the onset of sepsis, we assessed the numbers of blood and peritoneal-cavity circulating neutrophils and mononuclear cells 6 and 24 h after CLP. Although the increased numbers of blood circulating neutrophils and mononuclear cells were similar in Ido+/+ and Ido−/− mice, those of peritoneal-cavity circulating neutrophils and mononuclear cells were significantly higher in Ido−/− mice than in Ido+/+ mice (Table 1). Moreover, bacterial killing by the isolated neutrophils was similar between Ido+/+ and Ido−/− mice analyzed by a bactericidal-capacity assay (data not shown). These results suggest that the increase in recruited peritoneal-cavity circulating neutrophils and mononuclear cells may be involved in containment of the infection to the local focus and that Ido has an inhibitory effect on the recruitment of neutrophils and mononuclear cells, resulting in progression to sepsis.

TABLE 1.

Numbers of circulating neutrophils and mononuclear cells in Ido+/+ and Ido−/− mice after CLP

Location and cell type Time (h) No. of circulating cellsa
Ido+/+ mice
 Ido−/− mice
Sham operated CLP operated Sham operated CLP operated
Peritoneal cavity
    Neutrophils 6 0.024 ± 0.03 5.628 ± 4.96b 0.109 ± 0.09 18.758 ± 4.97b,c
24 0.109 ± 0.05 6.263 ± 2.19b 0.171 ± 0.07 17.633 ± 3.34b,c
    Mononuclear cells 6 0.607 ± 0.27 2.732 ± 0.72b 0.567 ± 0.74 8.261 ± 2.56b,c
24 1.315 ± 0.09 2.314 ± 0.65b 1.159 ± 0.14 7.866 ± 2.99b,c
Blood
    Neutrophils 6 0.125 ± 0.04 1.479 ± 0.95b 0.075 ± 0.04 3.162 ± 1.47b,c
24 0.156 ± 0.05 2.196 ± 0.74b 0.130 ± 0.03 3.829 ± 0.98b,c
    Mononuclear cells 6 4.925 ± 1.20 2.584 ± 2.46 3.137 ± 1.18 4.144 ± 1.88b,c
24 4.800 ± 0.87 2.073 ± 1.62 3.258 ± 0.86 3.652 ± 1.56b,c
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a

Ido+/+ and Ido−/− mice were subjected to sham or CLP surgery. Peritoneal exudate and blood samples were collected at 6 or 24 h after the surgery, and the neutrophils and mononuclear cells in the peritoneal cavity (106/cavity) and blood (106/ml) were counted. The data are expressed as means ± SD from 10 independent experiments.

b

P < 0.05 compared with sham-operated mice.

c

P < 0.05 compared with CLP-operated Ido+/+ mice using ANOVA.

CLP increased Ido mRNA expression in CD11b+ peritoneal cells.

We hypothesized that the Ido induced by CLP in peritoneal cells inhibits its recruitment because inhibition of Ido increased the recruitment of neutrophils and mononuclear cells to the peritoneal cavity after surgery. Indeed, expression of Ido mRNA was significantly increased in peritoneal cells isolated from Ido+/+ mice by CLP (Fig. 4A). Furthermore, Ido mRNA was expressed in the CD11b+ and CD11c+ cells isolated from the peritoneal cells after CLP (Fig. 4B). Specifically, most Ido+ peritoneal cells showed double staining for CD11b (Fig. 4C), suggesting that l-Kyn is mainly produced by CD11b+ cells among the other peritoneal cells. The number of CD11b+ cells in the peritoneal cavity was increased 6 h after CLP, and CD11b+ cells were induced to a greater extent by CLP in Ido−/− mice than in Ido+/+ mice (Fig. 4D). To examine how CLP increases Ido in peritoneal cells, the cultured peritoneal cells from mice were treated with LPS. LPS treatment increased Ido mRNA expression in the cultured peritoneal cells isolated from Ido+/+ mice (Fig. 4E). These results suggest that bacterial LPS induced by CLP increases Ido expression in the peritoneal CD11b+ cells and that increase of Ido activation may inhibit the recruitment of neutrophils and mononuclear cells to the infection focus.

FIG 4.

FIG 4

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Ido mRNA was increased in peritoneal cells by CLP. Ido+/+ and Ido−/− mice underwent CLP. The animals were humanly killed at the indicated times. (A) mRNA expression levels of Ido in peritoneal cells isolated from sham- or CLP-operated mice at 6 h after surgery were determined by quantitative real-time RT-PCR. (B) Cell fractionation was performed with the indicated antibodies and MACS magnetic beads from peritoneal cells isolated from sham- or CLP-operated mice 6 h after surgery. (C and D) Peritoneal cells were isolated from sham- or CLP-operated mice 6 h after surgery. Expression of CD11b (green), Ido (red), and DAPI nuclei (blue) in peritoneal cells was examined by immunofluorescent staining. The merged images are shown on the right. The CD11b+ cells were counted. (E) Peritoneal cells isolated from Ido+/+ or Ido−/− mice were treated or not with LPS (0.1 μg/ml) for 6 h, and mRNA expression levels of Ido were determined. The data are median IQR values from at least 5 independent experiments, with error bars indicating the range. *, P < 0.01 compared with Ido−/− mice; #, P < 0.05 compared with Ido+/+ CLP-operated mice; and †, P < 0.001 compared with sham-operated mice using ANOVA.

Ido deficiency in BM-derived cells with delayed mortality from CLP.

As described above, Ido in the peritoneal CD11b+ cells may be involved in increasing mortality rates after CLP. To explore the involvement of Ido in the BM-derived cells, we generated Ido chimeric mice by using a combination of irradiation and BMT. The Ido−/− BM-transplanted Ido+/+ mice (donor cells, Ido−/− BM cells; recipient animals, Ido+/+ mice) or Ido−/− mice (donor cells, Ido−/− BM cells; recipient animals, Ido−/− mice) showed higher survival rates following CLP than did the Ido+/+ BM-transplanted Ido+/+ mice (donor cells, Ido+/+ BM cells; recipient animals, Ido+/+ mice) or Ido−/− mice (donor cells, Ido+/+ BM cells; recipient animals, Ido−/− mice) (Fig. 5A). Serum l-Kyn levels significantly increased 6 h after CLP in Ido+/+ BM-transplanted Ido+/+ mice and Ido−/− mice, whereas CLP did not affect l-Kyn levels in the Ido−/− BM-transplanted Ido+/+ mice or Ido−/− mice (Fig. 5B). Thus, Ido in BM-derived cells, which include CD11b+ cells, is involved in the progression to sepsis after CLP.

FIG 5.

FIG 5

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Ido of bone marrow-derived cells is involved in mortality after CLP. Ido+/+ or Ido−/− bone marrow cells (5 × 106) were injected into lethally irradiated Ido+/+ or Ido−/− recipient mice. The chimeric mice were subjected to CLP 4 weeks after BMT. (A) Survival curves of animals after CLP. Statistically significant differences between the groups were determined using a log-rank test. The P values compared with Ido+/+ donor mice are shown. (B) The animals were humanely killed 6 h after surgery, and serum l-Kyn levels were measured. The data are means and SD from at least 5 independent experiments. *, P < 0.05 using ANOVA; ns, not significant.

Ido is involved in chemokine production at the infection focus.

To examine how Ido regulates recruitment of neutrophils and mononuclear cells to the site of infection, we measured the levels of chemotactic chemokines in Ido+/+ and Ido−/− mice after CLP. Expression levels of CXCL-2 and CXCL-1 mRNA in the peritoneal cells from Ido−/− mice were significantly higher than those from Ido+/+ mice 6 h after CLP (Fig. 6A), and the expression level of IL-17 mRNA in peritoneal cells from Ido−/− mice tended to increase compared to that from Ido+/+ mice. Similarly, serum CXCL-2 levels after CLP in Ido−/− mice were also higher than those in Ido+/+ mice (Fig. 6B). Moreover, the increase of CXCL-2 in Ido−/− mice after CLP was prevented by pretreatment with l-Kyn (Fig. 6B). In addition, LPS administration to the isolated cultured peritoneal cells increased CXCL-2 and CXCL-1 mRNA, and the increase was greater in cultured Ido−/− peritoneal cells than in Ido+/+ cells (Fig. 6C), suggesting that Ido induction by LPS has inhibitory effects against chemokine production by LPS in peritoneal cells. In addition to increased chemokine production by LPS in peritoneal cells, the number of CD11b+ cells in peritoneal cells was higher in Ido−/− mice, as described above (Fig. 4E). These findings suggest that Ido inhibits chemokine production in peritoneal cells due to synergistic effects of reduced productivity and cell numbers in its source. Indeed, LPS-induced CXCL-2 and CXCL-1 production and secretion into the medium were higher in peritoneal cells isolated from Ido−/− mice after CLP than in those from Ido+/+ mice (Fig. 6D). These results suggest that reduction of chemokine production in peritoneal CD11b+ cells occurs due to l-Kyn production by Ido induction after CLP and that it may reduce the recruitment of neutrophils and mononuclear cells to the peritoneal cavity.

FIG 6.

FIG 6

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Deficiency of Ido increased chemokines after CLP. (A to C) Ido+/+ and Ido−/− mice were subjected to CLP. The animals were humanly killed 6 h after surgery. (A) mRNA expression levels of CXCL-2, CXCL-1, and IL-17 in the isolated peritoneal cells were determined by quantitative real-time RT-PCR. (B) Serum CXCL-2 levels in the Ido+/+ or Ido−/− mice or l-Kyn-treated Ido+/+ or Ido−/− mice 6 h after surgery were quantified using ELISA. (C) Peritoneal cells were isolated from nontreated Ido+/+ or Ido−/− mice. The isolated cells were cultured for 2 h and were then treated or not with LPS (0.1 μg/ml) for the indicated periods of time. mRNA expression levels of CXCL-2 and CXCL-1 were determined by quantitative real-time RT-PCR. (D) Peritoneal cells were isolated from Ido+/+ or Ido−/− mice operated on with CLP 6 h after surgery. The isolated cells were cultured for 2 h and were then treated or not with LPS (0.1 μg/ml) for 6 h, and the CXCL-2 and CXCL-1 concentrations in the culture medium were measured by ELISA. The data are expressed as the median IQR from 6 independent experiments. *, P < 0.05 compared with Ido+/+ CLP mice; **, P < 0.01 compared with Ido+/+ mice or l-Kyn-treated Ido−/− mice; §, P < 0.01 compared with Ido−/− peritoneal cells, using ANOVA.

DISCUSSION

The present study investigated the contribution of Ido to the progression of sepsis due to CLP-induced bacterial peritonitis. The results indicate that Ido increases mortality rates through reduction of recruited neutrophils and mononuclear cells into the infection focus.

It has been reported that Ido plays a pivotal role in immune tolerance and that Ido is expressed in various types of cells, including astrocytes (17), epithelial and endothelial cells (18), tumor cells (19, 20), DCs (16), and macrophages (21, 22, 38). In CLP-induced bacterial peritonitis, Ido expression was increased in CD11b+ peritoneal cells, but not in neutrophils or NK cells, whereas its inhibition reduced mortality. LPS increased Ido expression in peritoneal cells in vitro, and Ido+ BM-derived cells were involved in increasing mortality rates. Thus, Ido expression in CD11b+ peritoneal cells has important roles in immune regulation in this model. Various functions of Ido for immune regulation have been reported. Activation of Ido in DCs suppresses T cell responses (39) and induces the generation of regulatory T cells (Tregs) via Trp metabolites in experimental autoimmune encephalomyelitis (40). Ido inhibits IL-17 production and promotes cytotoxic potential in mucosal NK cells during SIV infection (41). IFN-γ-induced Ido is involved in the suppression of Th17 in CIA, and the suppression of Th17 by IFN-γ was abolished with 1-MT (42). Furthermore, inhibition of Ido enhances T-cell response to influenza virus infection (43) and increases IL-10 production in BM-derived DCs (44).

In this study, we found that Ido decreased the number of CD11b+ peritoneal cells and the productivity of CXCL-2 and CXCL-1 in peritoneal cells. Chemokine production by LPS is regulated by NF-κB signaling (45, 46), and l-Kyn metabolites inhibit NF-κB activation by specifically targeting phosphoinositide-dependent protein kinase 1 (47). Thus, LPS stimulates both NF-κB and Ido, and the produced l-Kyn inhibits NF-κB, resulting in the reduction of CXCL-2 and CXCL-1 production. Indeed, pretreatment with l-Kyn prevented the increase of CXCL-2 and CXCL-1 production in Ido−/− mice. Moreover, pretreatment with l-Kyn reversed the mortality of Ido−/− mice. It is known that the downstream metabolites of Trp, including l-Kyn, suppress immune reactivity (28, 29). l-Kyn also functions as an endogenous ligand for the aryl hydrocarbon receptor, which modulates the functions of immune cells (48). In addition to the increased metabolites from Trp, Ido activation induces breakdown of Trp, which suppresses immune cell proliferation by reducing the availability of this essential amino acid under local tissue microenvironments (49). Although Ido depletion could explain the increase of CD11b+ cell recruitment in the peritoneal cavity via cell proliferation, the precise underlying mechanisms remain unclear, and further studies are needed to clarify these mechanisms.

It has been reported that Ido potentially has a dual role. Pathogens may reduce their growth by Ido-mediated tryptophan degradation (50). On the other hand, it may modulate immune cell recruitment and function and thus be detrimental to the host (32). Although Ido has both beneficial and disadvantageous effects in various cells, disadvantageous effects of Ido have been reported in immune cells (19–22, 51, 52). In this study, blockage of Ido in CD11b+ peritoneal cells reduced mortality after CLP, as well as increasing the rate of neutrophil and mononuclear cell recruitment into the infection focus. Similarly, another study found that 1-MT administration improves the survival rates of mice with sepsis induced by injection of cecal content (44).

As local infection progresses to sepsis, Ido shows further disadvantageous effects, including impaired endothelial function, decreased endothelial nitric oxide, and impaired immune functions (53). Recent studies have described Ido activity in the plasma as a prognostic factor in bacteremic patients and as a risk factor for posttraumatic sepsis (54, 55). There are many other pathways that produce leukocyte trafficking into the local focus. For example, granulocyte colony-stimulating factor (G-CSF) is known to cause extreme leukocytosis. A case of G-CSF-producing lung cancer with marked leukocytosis rapidly led to severe acute respiratory distress syndrome after pneumonia developed (56). Thus, inhibition of Ido may improve immune response to bacterial infection from the local infection focus to a systemic inflammatory response and may be more useful than other immunomodulatory strategies.

The experiment was restricted to a lethal model of CLP. It is therefore uncertain that Ido function would be the same in a subleathal model or with antibiotic therapy. Moreover, it is possible that the mechanism by which Ido functions in an intravenous-infection model is very different from the CLP model. Further studies are needed to resolve these uncertainties. In conclusion, we observed that Ido activation in peritoneal CD11b+ cells aggravated peritonitis and sepsis. Thus, blockade of Ido plays a critical role in host protection during bacterial peritonitis and sepsis.

ACKNOWLEDGMENTS

This work was supported by grants from the Uehara Memorial Foundation (to M.H.); a Grant-in-Aid for Research Activity Start-Up (24890270) from the Ministry for Education, Culture, Sports, Science and Technology of Japan (to M.H.); and the Takeda Science Foundation (to Y.O.), the SENSHIN Medical Research Foundation (to Y.O.), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to Y.O.), and the Ichiro Kanehara Foundation (to Y.O.).

Footnotes

Published ahead of print 11 August 2014

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