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a-Lactose Improves the Survival of Septic Mice by Blockade of TIM-3 Signaling to Prevent NKT Cell Apoptosis and Attenuate Cytokine Storm

Yao, Yao; Deng, Hai; Li, Pingfei; Zhang, Jian; Zhang, Junbo; Wang, Deping; Li, Songbo; Luo, Yixing; Wei, Zhengping; Bi, Guoyu; Yang, Xiang-Ping; Tang, Zhao-Hui

Author Information
doi: 10.1097/SHK.0000000000000717

Abstract

INTRODUCTION

Sepsis is a common and frequently fatal clinical disease that remains one of the leading causes of death among critically ill patients in the intensive care unit (1, 2). Patients with sepsis are systemically exposed to microbial agents that initiate a complex and dysregulated immune response (3). The host immune response to sepsis is characterized by a hyperinflammatory phase lasting for several days followed by a more protracted immunosuppressive phase. Yet recent studies have shown that both proinflammatory and anti-inflammatory responses occur early and simultaneously (4). There are multiple mechanisms involved in the development of immune disorder in sepsis (5). One of the important mechanisms is the sepsis-induced apoptosis of immune cells. Postmortem studies have shown extensive apoptosis of multiple types of immune cells, including CD4+ and CD8+ T cells, B cells, natural killer T (NKT) cells, and dendritic cells (DCs), occurs in all age groups (4, 6). Inhibition of immune cell apoptosis in mouse models by using genetic knockout animals, pharmaceutical inhibitors, or antiapoptotic cytokines improves survival in sepsis (4, 7, 8).

NKT cells express an invariant T-cell receptor (Va14-Ja18 in mouse and Va24-Ja18 in human) in addition to common natural killer (NK) cell markers. NKT cells play an important role in bridging innate and acquired immunity by rapidly responding to pathogens and secreting inflammatory cytokines to activate downstream effector immune cells (9–12). Data from different studies have shown contradictory findings of the role of NKT cells in sepsis: on one hand, NKT cell–derived inflammatory cytokines may contribute to the mortality of sepsis (12); on the other hand, NKT-deficient mice are more immunosuppressed after septic shock and have impaired survival (13). This could be due to the fact that NKT cells play distinct roles in different stages of the sepsis. So far the underlying mechanism by which NKT cells participate in the septic immune response and how their activity is regulated in sepsis remain to be further elucidated.

T-cell immunoglobulin and mucin domain 3 (Tim-3) is a type 1 transmembrane receptor and the expression of Tim-3 on immune cells including T cells, DCs, and macrophages is highly regulated in inflammation (14, 15). The natural ligand for Tim-3 is galectin-9, a β-galactoside–binding lectin (16). Cumulative findings indicate that the effect of Tim-3/galectin-9 interaction in immune regulation depends on the target cell type: in DCs, galectin-9 binding to Tim-3 leads to proinflammatory cytokine secretion (16, 17); by contrast, galectin-9 binding to Tim-3 induced Tim-3+ CD4+ T-cell apoptosis (16, 17). We previously showed that Tim-3/galectin-9 signaling plays an important role in regulating the function of NKT cells (18). In mouse model of nonalcoholic fatty liver disease (NAFLD), we found that Tim-3 is preferentially expressed on activated and proliferating NKT cells. Tim-3 expression was associated with elevated expression of inflammatory cytokines in NKT cells. Galectin-9 directly induced Tim-3+ NKT cell apoptosis by binding to Tim-3 and contributed to the depletion of NKT cells. Modulation of Tim-3/galectin-9 signaling significantly ameliorated liver inflammatory diseases in mouse model of NAFLD (18).

Here, we revealed that Tim-3 expression is upregulated on NKT cells in mouse model of sepsis and Tim-3+ NKT cells are more prone to apoptosis, resulting in the depletion of NKT cells. Blockade of the Tim-3/galectin-9 axis using α-lactose (galectin-9 antagonist) improved the survival rate of septic mice. Our data suggest that α-lactose could be a promising immunomodulatory agent in the treatment of sepsis.

METHODS

Mice and polymicrobial sepsis model

Adult wild-type (WT) C57BL/6 mice were purchased from Center for Experimental Animal of Wuhan University. The p35−/− with deletion of exons 1 and 2 of the interleukin (IL)-12 subunit p35 on a C57BL/6 background were obtained from The Jackson Laboratory (Bar Harbor, Maine). Polymicrobial sepsis was induced in mice by cecal ligation and puncture (CLP) as previously described (19, 20). In brief, mice (male, 8 weeks old) were anesthetized with ketamine/xylazine cocktail intraperitoneally (i.p.), followed by a midline abdominal incision (1.5–2 cm). The cecum was exposed, and ligated with a sterile silk suture 1 cm from the tip. It was perforated with 21G needle by single through-and-through puncture midway between the ligation and the tip of the cecum. After removing the needle, a small amount (droplet) of feces was extruded from both the mesenteric and antimesenteric penetration holes to ensure patency. The cecum was returned to the abdominal cavity and the incision was closed. Mice were resuscitated by injecting prewarmed saline (37°C, 1.25 mL/mice). Sham-operated mice were treated as described earlier with the exception of the ligation and puncture of the cecum. For α-lactose treatment, CLP mice were administered either 200 μL 5% or 10% α-lactose (Sigma-Aldrich, St. Louis, Mo) or 200 μL phosphate-buffered saline (PBS) by hypodermic injection into the loose skin around the neck at 0, 2, 6, 18, 36, and 72 h post-CLP. For lipopolysaccharide (LPS) treatment, WT C57BL/6 mice (male, 8 weeks old) were administered 200 μL LPS (20 mg/kg, Sigma-Aldrich) or PBS by i.p. injection. For the survival study, mice were constantly monitored for 28 days. All mice were maintained in a pathogen-free, temperature-controlled (20°C) facility with a 12-h light/dark cycle at the center for animal experiment of Tongji Medical College, Huzhong University of Science and Technology (HUST). All mice were given free access to food and water. All animal experiments fulfilled Tongji Medical College and HUST criteria for the humane treatment of laboratory animals and were approved by Tongji Medical College and HUST Animal Care and Use Committee.

Isolation and purification of MNCs, NKT cells, and DCs

Mononuclear cells (MNCs) were isolated from mouse liver or spleen as previously described (17, 18). NKT cells were purified from MNCs of mice by magnetic cell sorting (MACS) using mouse NKT cell isolation kits (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's instructions. Mouse immature DCs were isolated from mouse spleens as previously described (17, 18). After homogenization and centrifugation, DCs were purified from collagenase-treated mouse spleens by MACS with mouse DC isolation kit (Miltenyi Biotec). Purified DCs or NKT cells were resuspended in complete cell culture medium (RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 55 μM 2-mercaptoethanol). The purity of NKT cells and DCs in this experiment was above 90% determined by flow cytometry.

Flow cytometry analysis

For cell surface marker staining, the single-cell suspensions were stained with different anti-mouse antibodies, including CD3, CD4, CD8, NK1.1, Tim-3, CD11c, and annexin V (all from eBioscience, San Diego, Calif) as previously described (18). Intracellular cytokine staining was performed as previously described with anti-interferon (IFN)-γ (eBioscience) (18). Data were collected with a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ) and analyzed with FlowJo software (TreeStar, Ashland, Ore).

Quantification of immune cells

The total numbers of viable cells were determined using the Vi-CELL counter (Beckman Coulter, Fullerton, Calif) by trypan blue exclusion. Immune cells were also stained with fluorochrome-conjugated antibodies (eBioscience) to specific cell surface markers (NK1.1 and CD3 for NKT cells), and the percentage of cell subsets was determined by flow cytometry. The absolute cell counts for each subset population were calculated by using the following equation: count of subset = total viable cells count × percentage of subset/100.

Cell culture

NKT cells and DCs were cultured as previously described (17, 18). For Tim-3 expression assays, purified NKT cells (2 × 105) were resuspended in complete cell culture medium and were cultured with 0 ng/mL, 50 ng/mL, and 100 ng/mL recombination mouse IL-12 (rm-IL-12, eBioscience) for 8 h. Tim-3 expression in NKT cells was determined by flow cytometry. For NKT cell activation assays, purified NKT cells (2 × 105) were cocultured with or without purified DCs (5 × 105) in the presence or absence of 1 μg/mL LPS, in 24-well plates with or without 10 μg/mL anti–IL-12 monoclonal antibody (mAb, eBioscience) for 8 h. Tim-3 expression in NKT cells was determined by flow cytometry. For IL-12 secretion assays, purified mouse or human DCs (5 × 105) were preincubated with 40 μM α-lactose or with 40 μM sucrose for 1 h. Then, 3 μg/mL recombination mouse galectin-9 (Abcam, Cambridge, Mass) or LPS (1 μg/mL) was added to the culture medium; DCs were cultured for 48 h. The culture supernatants were collected and IL-12 production was determined by enzyme-linked immunosorbent assay (ELISA).

Enzyme-linked immunosorbent assay

Total liver protein was extracted as previously described (18). In brief, total liver protein was extracted from 20 mg liver tissue using 2 mL lysis buffer (Cell Signaling, Danvers, Mass) with protease inhibitors (Sigma-Aldrich). Total protein concentrations were determined with a BCA Protein Assay Kit (Pierce, Rockford, Ill). The concentrations of IL-12, IL-6, IL-10, and IFN-γ in mouse serum, liver tissue, or cell culture supernatants were quantified using mouse IL-12p70, IL-6, IL-10, or IFN-γ ELISA Ready-SET-Go Kits (eBioscience) according to the manufacturer's instruction.

Real-time RT-PCR

Total liver RNA was isolated and real-time reverse transcription polymerase chain reaction (RT-PCR) amplification was performed as previously described (18). In brief, complementary DNA (cDNA) was synthesized from 4 μg total RNA, using High-Capacity cDNA Reverse Transcription Kits (Invitrogen, Grand Island, NY). The primer sets for IL-15, Tim-3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Invitrogen. Quantitative PCR amplifications were performed using SYBR Green PCR Master Mix with the Prism 7900HT detection system (Applied Biosystems, Grand Island, NY). Gene expression levels of sample were normalized to GAPDH using ΔΔCt calculations.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5.0 software (GraphPad Software, Inc, San Diego, Calif ) and graph data are shown as mean ± standard deviation. Treatment-related differences were evaluated using the Student t test or analysis of variance. The relationships between two variables were evaluated by liner regression analysis. Survival curves were analyzed by the log-rank test. Statistical significance was set at a P value <0.05.

RESULTS

Increased Tim-3 expression and apoptosis in NKT cells during sepsis

To explore the potential functions of Tim-3 on NKT cells in sepsis, mice were subjected to CLP or sham surgery. After sepsis induction by CLP, we found that NKT cells are significantly enriched within the liver and a proportion of hepatic NKT cells expressed Tim-3 (Fig. 1A). This elevation could be detected after 4 h and was maintained for 10 days (Fig. 1B). The increased Tim-3 expression on NKT cells was also observed in spleen of CLP mice (data not shown). In addition, there is no significant difference in galectin-9 protein level in liver between CLP mice and sham mice at 12 and 24 h postoperative (data not shown).

Fig. 1
Fig. 1:
Elevated Tim-3 expression in NKT cells post-CLP.Mononuclear cells were obtained from the liver of WT mice subjected to CLP or sham treatment and stained with anti-mouse NK1.1, CD3, Tim-3, and annexin V. A, Representative dot plot of NK1.1 and Tim-3 staining in sham mice or CLP mice (gated on CD3+ NK1.1+ cell subset). B, Tim-3 expression in NKT cells was determined in liver of mice at different time points postoperation. C, Hepatic NKT cell apoptosis was determined at different time points post-CLP. D, Hepatic NKT cell apoptosis was determined in Tim-3+ or Tim-3 subset at 12 h post-CLP. E, IFN-γ expression of hepatic NKT cells in CLP mice versus sham mice. Results were expressed as the mean ± SD of seven independent experiments. Data were evaluated using Student t test or ANOVA. ANOVA indicates analysis of variance; CLP, cecal ligation and puncture; IFN, interferon; NKT, natural killer T; SD, standard deviation; Tim-3, T-cell immunoglobulin and mucin domain 3; WT, wild type.

Tim-3 has been shown to be able to mediate the apoptosis of NKT cells (18). To investigate this in CLP-treated animals, we measured apoptosis of NKT cells during sepsis. The percentage of apoptotic NKT cells was significantly increased within 48 h post-CLP (Fig. 1C), and most of the apoptotic NKT cells were Tim-3 positive (Fig. 1D). In line with the increased NKT apoptosis, the percentage of NKT cells in liver was significantly reduced in CLP mice compared with that in sham mice 24 h post-CLP (Supplemental Fig. 1A, http://links.lww.com/SHK/A449). Using annexin V as a marker of apoptosis, we found that there was significant correlation between apoptosis and expression of Tim-3 in NKT cells from CLP mice (Supplemental Fig. 1B, http://links.lww.com/SHK/A449).

Rapid secretion of IFN-γ by NKT cells amplifies the immune response by activating other immune cells and resulting in induction of other proinflammatory cytokines in sepsis (11, 12, 21). We found that more NKT cells in CLP mice express IFN-γ compared with those in sham mice (Fig. 1E). Furthermore, in CLP mice, the Tim-3+ NKT cell subset significantly produced more IFN-γ compared with the Tim-3 NKT cell subset (Supplemental Fig. 1C, http://links.lww.com/SHK/A449).

IL-12 secreted by LPS-stimulated DCs enhances Tim-3 expression on NKT cells in sepsis

Bacterial endotoxins, such as LPS, are potent inducers of inflammatory cytokines in immune cells, which can trigger life-threatening cytokine storm in sepsis (22). We wondered if LPS is involved in the induction of Tim-3 expression in NKT cells. Lipopolysaccharide or PBS was injected i.p. into mice, and then Tim-3 expression in NKT cells was examined. Tim-3 expression in hepatic NKT cells was significantly increased in LPS-challenged mice compared with that in PBS control group (Fig. 2A). Furthermore, in LPS-challenged mice, the expression of Tim-3 in NKT cells shared a similar pattern with CLP mice within 24 h (Fig. 1B). These findings suggested that LPS might be involved in inducing Tim-3 expression on NKT cells post-CLP. However, it has been shown that NKT cells do not directly respond to LPS stimulation in vitro (23, 24). To test if LPS can directly induce Tim-3 expression in NKT cells, NKT cells were stimulated with LPS for 8 h and we found no difference of Tim-3 expression after LPS stimulation, suggesting that LPS indirectly induces the Tim-3 expression on NKT cells (data not shown).

Fig. 2
Fig. 2:
IL-12 secreted by DCs induces Tim-3 expression on NKT cells postsepsis.NKT cells and DCs were purified from the spleen and liver of mice using MACS. A, Tim-3 expression in hepatic NKT cells was determined in mice injected with LPS (20 mg/kg) or PBS. B and C, Purified NKT cells from WT mice were cocultured with DCs in the presence of LPS (1 μg/mL) or PBS for 8 h (B), or were cultured in the presence of rm-IL-12 (50 ng/mL, 100 ng/mL) or PBS for 8 h (C), and then Tim-3 expression on NKT cells was evaluated. D and E, IL-12 KO mice and WT mice were subjected to CLP, and then Tim-3 expression on hepatic NKT cells (D) and apoptosis of hepatic NKT cells (E) were evaluated at 8 h post-CLP. F, Purified NKT cells from WT mice were cocultured with DCs deriving from IL-12 KO mice or WT mice, and then Tim-3 expression on NKT cells in response to LPS (1 μg/mL) stimulation was evaluated. Results were mean ± SD of five to seven independent experiments. Data were evaluated using Student t test or ANOVA. ANOVA indicates analysis of variance; CLP, cecal ligation and puncture; DC, dendritic cell; IL, interleukin; KO, knockout; MACS, magnetic cell sorting; LPS, lipopolysaccharide; NKT, natural killer T; PBS, phosphate-buffered saline; rm-IL-12, recombination mouse IL-12; SD, standard deviation; Tim-3, T-cell immunoglobulin and mucin domain 3; WT, wild type.

Our previous work suggested that Tim-3 is a functional activated marker in NKT cells (18). It has been shown that NKT cell activation in sepsis is induced by IL-12, which is produced by LPS-stimulated DCs (23). We wondered if the induction of Tim-3 expression in NKT cells by LPS in vivo is mediated by IL-12. To test this, NKT cells were stimulated with LPS in the presence of DCs for 8 h. When cocultured with DCs, Tim-3 expression was upregulated in NKT cells after addition of LPS (Fig. 2B) and the levels of IL-12 were significantly elevated in the supernatants from the cocultured cells treated with LPS compared with those in the control group (data not shown).

To establish a direct effect of IL-12 on Tim-3 expression in NKT cells, we stimulated the NKT cells in the presence or absence of recombinant mouse interleukin-12 for 8 h. We found that IL-12 strongly induced Tim-3 expression in NKT cells (Fig. 2C). By contrast, other IL-12 family cytokines, including IL-23 and IL-27, had no effect on Tim-3 expression in NKT cells (data not shown). Administration of a neutralizing antibody against IL-12 attenuated LPS-induced Tim-3 expression in NKT cells in the presence of DCs, suggesting that LPS induced Tim-3 expression via the induction of IL-12 in DC cells (Supplemental Fig. 2A, http://links.lww.com/SHK/A450). Consistent with this, CLP treatment induced significantly higher amount of IL-12 in the liver tissue at 4 and 8 h post-CLP (data not shown).

Next, we determined that the Tim-3 expression level in IL-12–deficient (Il12p35−/−) mice induced sepsis by CLP. At 8 h post-CLP, the percentage of Tim-3+ NKT cells was significantly reduced in Il12p35−/− mice compared with that in WT mice (Fig. 2D). This was associated with a significant reduction in apoptosis of NKT cells accompanied by a significantly increased percentage of NKT cells in the liver of Il12p35−/− mice (Fig. 2E; Supplemental Fig. 2B, http://links.lww.com/SHK/A450). When NKT cells were cocultured with DCs from Il12p35−/− mice, the expression level of Tim-3 in NKT cells in response to LPS stimulation was significantly decreased compared with that in NKT cells cocultured with DCs from WT mice (Fig. 2F), suggesting that LPS induces Tim-3 expression in NKT cells via the production of IL-12 by DCs.

α-Lactose prevents NKT cell apoptosis and reduces Tim-3 expression during sepsis

We previously showed that homeostatic maintenance of NKT cells could significantly ameliorate liver inflammatory disease by exogenous application of recombinant human galectin-9 (18). We wondered if manipulating the Tim-3/galectin-9 axis would attenuate the severity of sepsis in the CLP model. Compared with control PBS group, administration of recombinant mouse galectin-9 (rm-galectin-9) had no effect on the survival rate of CLP mice (data not shown). A recent study indicated that administration of anti–Tim-3 mAb exacerbated the uncontrolled inflammation and led to reduced survival rate in CLP mice (25). α-Lactose binds to the carbohydrate-binding domain of galectin-9 and limits its engagement with Tim-3 (16). We used α-lactose to modulate the galectin-9/Tim-3 signaling axis in the CLP mouse model. Cecal ligation and puncture–mice treated with α-lactose showed a reduced rate of hepatic NKT cell apoptosis at 8 and 24 h post-CLP (Fig. 3A), which is consistent with our previous in vitro findings that α-lactose inhibited galectin-9–induced NKT cell apoptosis (18). The absolute number (Fig. 3B) and the percentage of hepatic NKT cells (Supplemental Fig. 3A, http://links.lww.com/SHK/A451) are also increased after α-lactose treatment. Similar phenomenon has been observed in spleen and bone marrow of mice treated with α-lactose (data not shown). Moreover, administration of α-lactose reduced the percentage of Tim-3+ NKT cell subset and the mRNA amount of Tim-3 in hepatic NKT cells (Fig. 3C; Supplemental Fig. 3B, http://links.lww.com/SHK/A451).

Fig. 3
Fig. 3:
α-Lactose prevents NKT cell apoptosis and reduces Tim-3 expression.WT mice were subjected to CLP or sham surgery, and then CLP mice were administered α-lactose (10%, 200 μL) or PBS (200 μL) by hypodermic injection at 0, 2, 6, 18, and 36 h post-CLP. NKT cells were purified using MACS. A and B, Hepatic NKT cell apoptosis (A) and the absolute NKT cell number in liver (B) were assayed in CLP mice and sham mice by flow cytometry at 8 or 24 h postoperation. C, Hepatic Tim-3+ NKT cell subset was determined in CLP mice treated with α-lactose or PBS at 8 and 24 h post-CLP. Results were mean ± SD of seven independent experiments. Data were evaluated using ANOVA. ANOVA indicates analysis of variance; CLP, cecal ligation and puncture; MACS, magnetic cell sorting; NKT, natural killer T; PBS, phosphate-buffered saline; SD, standard deviation; Tim-3, T-cell immunoglobulin and mucin domain 3; WT, wild type.

α-Lactose attenuates sepsis-induced cytokine storm

The rapid release of both proinflammatory and anti-inflammatory cytokines is a core feature during sepsis. We assessed a panel of inflammatory cytokine expression in CLP mice treated with α-lactose. We found significantly decreased serum IFN-γ level in CLP mice treated with α-lactose versus PBS (Fig. 4A). The proportion of IFN-γ+ NKT cells was decreased in liver of CLP mice treated with α-lactose (Fig. 4B), which is consistent with our earlier finding that administration of α-lactose reduced the percentage of Tim-3+ NKT cells. Several studies have provided valuable insights into the early potent secretion of IFN-γ by NKT cells that activates other immune cells and results in the inflammatory cytokine cascade in sepsis (12, 21, 26). Therefore, we next assessed the effect of α-lactose on other cytokines IL-6 and IL-10. Treatment with α-lactose led to reduced serum IL-6 and IL-10 levels in CLP mice (Fig. 4, C and D). We also assessed the expression of IL-15, which is a crucial factor mediating the proliferation of NKT. The IL-15 mRNA expression was decreased in liver of CLP mice treated with α-lactose (Fig. 4E).

Fig. 4
Fig. 4:
α-Lactose attenuates sepsis-induced cytokine storm.WT mice were subjected to CLP or sham surgery, and then CLP mice were administered α-lactose (10%, 200 μL) or PBS (200 μL) by hypodermic injection at 0, 2, 6, 18, and 36 h post-CLP. A, C-D, The serum levels of IFN-γ (A), IL-6 (C), and IL-10 (D) were evaluated in CLP mice treated with α-lactose or PBS by ELISA at 8 and 24 h post-CLP. B, IFN-γ expression in hepatic NKT cells was determined in CLP mice treated with α-lactose or PBS by intracellular cytokine staining at 8 h post-CLP. E, The mRNA expression of IL-15 in liver was evaluated in sham mice or CLP mice treated with α-lactose or PBS by real-time PCR at 8, 24, and 48 h postoperation. Results were mean ± SD of seven independent experiments. Data were evaluated using Student t test or ANOVA. ANOVA indicates analysis of variance; CLP, cecal ligation and puncture; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IFN, interferon; IL, interleukin; NKT, natural killer T; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; SD, standard deviation; WT, wild type.

α-Lactose inhibits galectin-9–induced IL-12 production by DCs

Because α-lactose competitively inhibited galectin-9–induced Tim-3+ NKT cell apoptosis and Tim-3+ NKT cell subset secreted more IFN-γ (18), we are puzzled why administration of α-lactose reduced the percentage of Tim-3+ NKT cell subset and IFN-γ secretion post-CLP. Given the above-mentioned finding that IL-12 production by LPS-stimulated DCs upregulated Tim-3 expression on NKT cells, we wondered if administration of α-lactose concurrently inhibits IL-12 production resulting in downregulation of Tim-3 expression on NKT cells. Indeed, our findings indicated that IL-12 production was significant reduced in serum and liver of CLP mice after treatment with α-lactose (Fig. 5, A and B). However, when NKT cells were cocultured with LPS in the presence of DCs, the addition of α-lactose did not alter Tim-3 expression and IL-12 production, suggesting that α-lactose indirectly affects the Tim-3 expression in NKT cells (data not shown). Inspired by previous studies that galectin-9 is able to synergize with LPS in IL-12 production of DCs (27), we hypothesized that this contradiction could be due to α-lactose blocking the effect of galectin-9–induced IL-12 production in DCs. To test this, we measured the effect of α-lactose on rm-galectin-9–induced IL-12 production in DCs and found that α-lactose significantly inhibited IL-12 secretion (Fig. 5C). The addition of a Tim-3–blocking antibody (clone RMT3-23) also inhibited rm-galectin-9–induced IL-12 production in DCs (Fig. 5C), suggesting that α-lactose, similar to Tim-3–blocking antibody, reduces IL-12 production in DCs by blockade of galectin-9 binding to Tim-3.

Fig. 5
Fig. 5:
α-Lactose inhibits galectin-9–induced IL-12 production by DCs.WT mice were subjected to CLP, and then CLP mice were administered either α-lactose (10%, 200 μL) or PBS (200 μL) by hypodermic injection at 0, 2, 6, and 18 h post-CLP. NKT cells and DCs were purified from the spleen or liver of WT mice by MACS. A and B, IL-12 level in liver tissue (A) or serum (B) was determined by ELISA in CLP mice treated with α-lactose or PBS at different time points post-CLP. C, Purified mouse DCs were preincubated with α-lactose (40 μM) or sucrose (40 μM), anti–Tim-3 Abs (30 μg/mL), or IgG (30 μg/mL) for 1 h, and then cultured in the presence of rm-galectin-9 (3 μg/mL) or LPS (1 μg/mL, positive control) for another 48 h. The amount of IL-12 in culture supernatants was determined by ELISA. Results were mean ± SD of five to seven independent experiments. Data were evaluated using ANOVA. ANOVA indicates analysis of variance; CLP, cecal ligation and puncture; DC, dendritic cell; ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; IL, interleukin; LPS, lipopolysaccharide; MACS, magnetic cell sorting; NKT, natural killer T; PBS, phosphate-buffered saline; rm-galectin-9, recombinant mouse galectin-9; SD, standard deviation; Tim-3, T-cell immunoglobulin and mucin domain 3; WT, wild type.

α-Lactose improves the survival of CLP mice

The beneficial effect of α-lactose on survival in sepsis was tested in the CLP model. Survival was recorded for 28 days. Administration of α-lactose significantly reduced the mortality rate of CLP mice (n = 30) to 16%, compared with 63% in control group (n = 30) at 72 h post-CLP (P < 0.01) (Fig. 6). The CLP mice treated with α-lactose that survived more than 7 days did not show further mortality until 28 days post-CLP (Fig. 6). Overall, the 28-day mortality rate was 20% in mice treated with α-lactose, compared with 73% in the control group (P < 0.01) (Fig. 6). Increasing the dose of α-lactose (10%) led to a significantly greater protective effect in CLP mice over the lower concentration (5%, P < 0.05) (Fig. 6), which is consistent with our previous finding that α-lactose inhibited galectin-9–induced NKT cell apoptosis in a dose-dependent manner (18). Sucrose is a related disaccharide to α-lactose, but 10% sucrose had no beneficial effect on survival of CLP mice (Fig. 6).

Fig. 6
Fig. 6:
α-Lactose improves the survival of CLP mice.WT mice were subjected to CLP. Survival curve of CLP mice administered α-lactose (5% or 10%, 200 μL), sucrose (10%, 200 μL), or PBS (200 μL) by hypodermic injection at 0, 2, 6, 18, 36, and 72 h post-CLP. Survival was recorded for 28 days. Data were analyzed using the log-rank test (n = 30 per group). CLP indicates cecal ligation and puncture; PBS, phosphate-buffered saline; WT, wild type.

DISCUSSION

Two immunopathologic hallmarks of sepsis are cytokine storm and immunosuppression. Natural killer T cells are involved in not only the initial hyperinflammatory phase but also the protracted immunosuppressive phase during sepsis. Activated NKT cells and their early potent IFN-γ secretion act as a primary initiator to rapidly activate other immune cell types, such as macrophages, DCs, NK cells, and effector T cells, and then to ignite inflammatory cascade in the initial phase of sepsis (11, 12). Subsequently, sepsis-induced apoptosis led to severe depletion of NKT cells accompanied with decrease of IFN-γ release, which was thought to significantly promote immunosuppression, a major cause of death in sepsis (10, 26).

Our previous study indicated that Tim-3 is preferentially expressed in activated or proliferative NKT cells and Tim-3+ NKT cells produce more IFN-γ (18). In the present study, we found that Tim-3 expression in NKT cells is rapidly enhanced in a mouse sepsis model, peaked at 24 h, and increased around 7-fold in CLP mice. After sepsis induction, the elevated Tim-3 expression in NKT cells correlated with the more IFN-γ secretion by Tim-3+ NKT cell subset. Drastically increased expression of Tim-3 may be a marker of overly activated NKT cells after sepsis onset. Therefore, our data were consistent with previous finding that overactivation of NKT cells is potential deleterious effects of sepsis, although early activation of NKT cells is beneficial to pathogenic microbe clearance.

Our data were consistent with the previous finding that NKT cell apoptosis is prominently increased post-sepsis, resulting in the significant depletion of NKT cells. We found that there is significant correlation between elevated Tim-3 expression and increased NKT cell apoptosis. We found that there is no significant difference in galectin-9 protein level between CLP mice and sham mice postoperative. Thus, overexpression of Tim-3 in NKT cells contributed to increased NKT cell apoptosis and depletion, which ultimately might be an important cause of immunosuppression during the later stages of sepsis.

Our data argue that Tim-3 can serve as a marker of activated NKT cells after the onset of sepsis. Elucidating the mechanisms underlying the induction of Tim-3 on NKT cells could help us to provide new strategies for modulating the functions of NKT cells in sepsis. Although we found a positive correlation between endotoxin (LPS) levels and the expression levels of Tim-3 on NKT cells in vivo, NKT cells do not directly respond to LPS (23, 24), suggesting that LPS might induce Tim-3 expression indirectly. Interleukin-12 is an important immune mediator induced by LPS in DCs and it plays a vital role for the resistance against various infectious agents (28, 29). Here, we showed that IL-12 secreted by DCs on exposure to LPS strongly induced Tim-3 expression on NKT cells. Interleukin-12 is known to induce T-bet expression through signal transducer and activator of transcription 4 and T-bet has been shown to be able to partly induce Tim-3 expression in Th1 cells (30, 31).

α-Lactose, a disaccharide sugar, is able to compete with galectin-9 for the binding to Tim-3 (16). Our previous study indicated that α-lactose in vitro competitively inhibited galectin-9–induced apoptosis of NKT cells via Tim-3/galectin-9 signaling in a dose- and time-dependent manner (18). Here, we identified a beneficial effect of α-lactose in a mice model of sepsis. The 28-day mortality rate of CLP mice treated with α-lactose was significantly less than that of the PBS-treated control group. The protective effect of α-lactose was correlated with decreased apoptosis and depletion of NKT cells in liver, spleen, and bone marrow. Interestingly, we found that treatment of α-lactose leads to a reduced expression of IL-15, which is a key factor for the proliferation of NKT cells. Our data suggested that the increased hepatic NKT cell population could not be attributed to the enhanced proliferation mediated by pro-proliferative cytokines such as IL-15 or migration of NKT cells from other tissues, but due to the reduced apoptosis of NKT cells. We deduced that administration of α-lactose to block proapoptotic effect of galectin-9 may play a crucial role in recovering NKT cell subset after sepsis.

Administration of α-lactose not only inhibited the apoptosis of Tim-3+ NKT cells but also suppressed Tim-3 expression in NKT cells by blocking galectin-9–induced IL-12 production in DCs after CLP treatment. Subsequently, IFN-γ+ NKT cell subset and IFN-γ level were simultaneously decreased within 24 h, which is consistent with Tim-3+ NKT cells secreting more IFN-γ. Interferon-γ plays a major role in activating both innate and acquired immune cells and resulting to facilitate inflammatory cytokine storm in the initial phase of sepsis (11). Administration of α-lactose also downregulated expression levels of both proinflammatory and anti-inflammatory cytokines, including IFN-γ, IL-6, and IL-10, to attenuate overwhelming production of inflammatory cytokines induced by sepsis.

At the present, a lot of immunomodulatory agents to sepsis are pluripotent cytokine, which have been assumed to potentially enhance the deleterious aspects of anti-inflammation or proinflammation (5). Our data suggested that α-lactose might represent a novel, convenient, and inexpensive immunotherapeutic agent to sepsis with multiple advantages: α-lactose is a disaccharide sugar derived from galactose and glucose, neither inherently proinflammatory nor anti-inflammatory. α-Lactose is a common pharmaceutical excipient in more than 2,300 oral capsule and tablet formulations approved by the Food and Drug Administration (32). It can be used in intravenous injections (32). Adverse reactions to α-lactose are minor. Further investigation about the influence of α-lactose on immune responses during sepsis in clinical settings is warranted.

Acknowledgments

We thank Dr Arian Laurence (Translational Gastroenterology Unit, Experimental Medicine Division, John Radcliffe Hospital, University of Oxford) for critical reading of the manuscript. We thank Prof. Youhua Hao, Fan Zhu, and Honghui Ding at Division of Clinical Immunology, Tongji Hospital, for the assistance in flow cytometry. We thank the staff at Division of Animal Experiment Center, Tongji Hospital.

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Keywords:

Apoptosis; α-lactose; natural killer T cells; sepsis; T-cell immunoglobulin and mucin domain 3; DC; dendritic cell; NAFLD; nonalcoholic fatty liver disease; NK; natural killer; NKT; natural killer T; Tim-3; T-cell immunoglobulin and mucin domain 3

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