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. Author manuscript; available in PMC: 2014 Apr 1.
Published in final edited form as: Immunobiology. 2012 Jul 27;218(4):561–569. doi: 10.1016/j.imbio.2012.07.022

Natural killer T cells are required for lipopolysaccharide-mediated enhancement of atherosclerosis in apolipoprotein E-deficient mice

Yasuhiro Andoh 1,2,@, Hisako Ogura 1,@, Masashi Satoh 1,3, Kentaro Shimano 3, Hironori Okuno 3, Satoshi Fujii 4, Naoki Ishimori 2, Koji Eshima 3, Tatsuro Otani 3, Yukihito Nakai 2, Luc Van Kaer 5, Hiroyuki Tsutsui 2, Kazunori Onoé 1, Kazuya Iwabuchi 1,3,*
PMCID: PMC3535521  NIHMSID: NIHMS405084  PMID: 22954709

Abstract

Lipopolysaccharide (LPS) has been shown to accelerate atherosclerosis and to increase the prevalence of IL-4-producing natural killer T (NKT) cells in various tissues. However, the role of NKT cells in the development of LPS-induced atherosclerotic lesions has not been fully tested in NKT cell-deficient mice. Here, we examined the lesion development in apolipoprotein E knockout (apoE-KO) mice and apoE-KO mice on an NKT cell-deficient, CD1d knockout (CD1d-KO) background (apoE-CD1d double knockout; DKO). LPS (0.5 μg/g body weight/wk) or phosphate-buffered saline (PBS) was intraperitoneally administered to apoE-KO and DKO mice (8 wk old) for 5 wk and atherosclerotic lesion areas were quantified thereafter. Consistent with prior reports, NKT cell-deficient DKO mice showed milder atherosclerotic lesions than apoE-KO mice. Notably, LPS administration significantly increased the lesion size in apoE-KO, but not in DKO mice, compared to PBS controls. Our findings suggest that LPS, and possibly LPS-producing bacteria, aggravate the development of atherosclerosis primarily through NKT cell activation and subsequent collaboration with NK cells.

Keywords: NKT cells, NK cells, CD1d, Inflammation, Atherosclerosis

Introduction

Atherosclerosis is recognized as an inflammatory vascular disease that involves both the innate and acquired immune systems (Binder et al. 2002, Hansson et al. 2006). Thus, various components of the immune system are likely to be involved as genetic or environmental factors that promote or regulate development of atherosclerosis. Using atherosclerosis-prone mice, such as apoE knockout (apoE-KO) (Piedrahita et al. 1992) or low-density lipoprotein receptor knockout (LDLR-KO) mice (Ishibashi et al. 1993), the roles of various components in innate and acquired immunity as pro- or anti-atherogenic factors have been extensively examined (Hansson et al. 2006) in the hope to identify novel preventive and therapeutic targets.

Natural killer T (NKT) cells represent a unique subset of the T cell lineage, which simultaneously expresses markers of natural killer cells (Van Kaer 2007). NKT cells recognize various lipid antigens in the context of CD1d molecules (Gumperz et al. 2000) such as the marine sponge-derived glycosphingolipid α-galactosylceramide (α-GalCer) (Kawano et al. 1997). Activated NKT cells can produce interferon (IFN)-γ, interleukin (IL)-4, and osteopontin (Arase et al. 1993; Arase et al. 1996; Diao et al. 2004), each of which has possible pro-atherogenic properties (Gupta et al. 1997; King et al. 1997; Matsui et al. 2003). Accordingly, others and we have reported that NKT cells accelerate atherogenesis in various mouse models (Tupin et al. 2004; Nakai et al. 2004; Major et al. 2004; Aslanian et al. 2005; VanderLaan et al. 2007; Rogers et al. 2008; To et al. 2009).

It has been well documented that microbial infections enhance the development of atherosclerotic lesions in both human and animal models (Hansson et al. 2006). Indeed, the ligands for toll-like receptors (TLR) and the associated TLR signaling pathway have multiple roles in the development of atherosclerosis (Björkbacka 2006; Erridge 2009). In LPS-treated mice, an exaggerated lesion development and a concomitant increase of IL-4-producing NKT cells was detected in peripheral blood, liver, spleen, thymus and atherosclerotic plaques in a mouse model (Ostos et al. 2002) However, the direct relationship between endotoxin-mediated aggravation of atherosclerosis and the functions of NKT cells remains to be examined.

In the present study, using atherosclerosis-prone apoE-KO mice and apoE-KO mice that carry a disruption in the CD1d gene (apoE-CD1d double KO; DKO) and therefore lack NKT cells, we were able to directly examine the involvement of NKT cells in LPS-induced exacerbation of atherosclerosis. Furthermore, we demonstrate the collaboration of NKT cells with NK cells, another subset of innate lymphocytes, for the LPS-induced exacerbation of atherosclerosis in apoE-KO mice. The involvement of bacterial lipids in exacerbating lesion development in atherosclerosis in mice is discussed as an experimental model of human pathology.

Materials and Methods

Mice

Female apoE-KO mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and DKO mice were established by mating CD1d-KO and apoE-KO mice at the Center for Disease Models, Institute for Genetic Medicine, Hokkaido University. Both strains were on the C57BL/6 genetic background. In some experiments, NK cells were depleted in apoE-KO mice with-asialoGM1 antibody (Suzuki et al. 1985). In brief, a dose of 50 μl of anti-asialo-GM1 antiserum (Wako Pure Chemical Industries, Ltd., Japan) diluted in 250 μl of pyrogen-free phosphate buffered-saline (PBS) was intravenously injected 1 day before treatment with LPS. These mice were fed a regular chow diet. Animal care and experimental procedures conformed to the regulations of the Committees of Animal Experimentation at Hokkaido University and Kitasato University School of Medicine.

Induction of atherosclerotic lesions

ApoE-KO mice (8 wk old) were divided into 2 groups: one group received weekly intraperitoneal (i.p.) injections of LPS (from E. coli O55: B5′, Sigma Chemical Co., Sr. Louis, MO) at 0.5 μg body weight (BW) for 5 wk; another group received PBS alone. DKO mice were also divided into 2 groups, and administered either LPS or PBS in the same manner as for apoE-KO mice. At 13 wk of age, mice were sacrificed for analysis.

Blood chemistry

Total cholesterol (T-cho), high-density lipoprotein (HDL) cholesterol (HDL-cho), triglyceride (TG), glutamic-pyruvic transaminase (GPT), and fasting blood sugar (FBS) concentrations in sera were quantified by colorimetric assays with Fuji Drychem System (Fujifilm Medical, Osaka, Japan) according to the manufacturer’s protocol, as described elsewhere (Nakai et al. 2004).

Quantitative analyses of atherosclerotic lesion areas

Atherosclerotic lesions were analyzed as previously described (Nakai et al. 2004). In brief, the basal portion of the heart and proximal aortic root were excised, embedded in OCT compound and frozen in liquid nitrogen. Eight serial cryosections of 10 μm-thickness at 80 μm intervals throughout the aortic sinus were stained with Oil red O (Sigma) and hematoxylin. The lesion areas were quantified by computerized image analysis system (Scion Image software, Scion Corp., Frederick, MD). Elastica Masson staining was performed to analyze the composition of the lesion using 3 aortic cross-sections per animal from 10 animals. The ratio of collagen-rich matrix areas versus cell-rich areas was defined in each group of mice.

Flow cytometry

Splenocytes were prepared by teasing spleen with a glass homogenizer and red blood cells were lysed with Tris-NH4Cl solution. Hepatic mononuclear cells (HMNC) were isolated from liver homogenates by density-gradient centrifugation with 33% Percoll (GE Healthcare Life Sciences, Piscataway, NJ) as previously reported (Watanabe et al. 1992). The cells were incubated with 2.4G2 monoclonal antibody (mAb) (anti-FcγRIII/II) to block non-specific binding of primary mAb, and then reacted with phycoerythrin (PE)-conjugated mouse CD1d-tetramer (Medical Biological Laboratory, Takatou, Japan) loaded with α-GalCer (α-GalCer-CD1d-tetramers) according to the manufacturer’s protocol (Nakai et al. 2004). After washing, cells were stained with a combination of the following fluorescently labeled mAbs: fluoresceine isothiocyanate (FITC)-anti-TCRβ chain (H57-597) and allophycocyanin (APC)-anti-NK1.1 (PK136)(BD Bioscience, San Jose, CA). Stained cells were acquired with a FACSCalibur™ flow cytometer (BD Bioscience) and analyzed with CellQuest software (BD Bioscience Immunocytometry Systems). Propidium iodide (Sigma) positive cells were electronically gated out from the analysis.

Quantification of serum cytokines

The concentrations of various cytokines in sera were quantified by Cytometric Bead Array (CBA; BD Biosciences) according to the manufacturer’s protocol. Th1/Th2 and inflammatory cytokines, including IFN-γ, tumor necrosis factor (TNF)-α, IL-2, -4, -5, -6, -12p70 and monocyte chemoattractant protein (MCP)-1 were quantified with the bead-based flow cytometric method in sera obtained at 1 wk or over time after the last LPS administration.

Collection and culture of peritoneal exudate cells (PECs)

PECs were elicited by intraperitoneal injection of 4.05% thioglycollate and harvested by washing the peritoneal cavity of mice with 15 ml of cold PBS as previously described (Ato et al. 1999/2000). Collected peritoneal cells (5 × 105 cells/well) were incubated with RPMI-1640 medium supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 μg/ml) in the presence of LPS (0.1 or 1 μg/ml at final concentration). In some experiments, PECs were co-cultured with 5 × 104 NKT hybridoma (2E10) cells (Nyambayar et al. 2007). Culture supernatants were harvested and cytokines were quantified with Mouse Th1/Th2 10plex FlowCytomix™ Multiplex (Bender MedSystems GmbH) according to the manufacturer’s protocol, using flow cytometry (Satoh et al. 2012).

Intracellular staining and analysis of cytokines

Two hours after either α-GalCer or LPS injection into WT mice, spleen cells or HMNC were prepared. The cells were cultured for 4hr in the presence of brefeldin A and stained stained with APC-anti-NK1.1 and FITC-anti-TCRβ mAb, followed by fixation with paraformaldehyde. Then, the cells were permeabilized, stained with PE-anti-IFN-γ or PE-anti-IL-4 mAb, and analyzed for fluorescence intensity of anti-cytokine mAbs in the NKT cell-gated (NK1.1+/TCRβ+) population as previously described (Minami et al. 2005).

Statistical analysis

Results are shown as means ± standard errors (SE). Statistical analysis was performed by either Student’s t-test or the Mann-Whitney U rank sum test. All data analyses were performed using StatView software (Abacus Concept, Berkeley, CA). Values with P < .05 were considered to be statistically significant.

Results

Impact of LPS on atherosclerosis in ApoE-KO mice lacking NKT cells

To examine the potential role of NKT cells in the exacerbation of atherosclerosis mediated by LPS, we analyzed the lesion areas in apoE-KO and DKO mice that had received weekly administrations of LPS (0.5 μg/g BW) for 5 wk duration. This dose of LPS was selected based on a previous report (Ostos et al. 2002) (50 μg/mouse; about 2.5 μg/g BW) and our preliminary experiments, which did not induce overt clinical manifestations of sepsis syndrome/septic shock. DKO mice injected with PBS developed significantly smaller lesion areas compared to similarly treated apoE-KO mice (301,545 ± 43,198 μm2 versus 149,511 ± 20,113 μm2, p = 0.0114)(Fig. 1A, B). These results are consistent with previous reports (Tupin et al. 2004; Major et al. 2004).

Figure 1. Atherosclerotic lesion areas in apoE-KO and DKO mice treated with LPS.

Figure 1

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(A) Representative histology of the atherosclerotic lesions from apoE-KO and apoE-KO/CD1d-KO (DKO) mice treated with LPS or PBS. Arrows represent the oil red O-positive, atherosclerotic lesions typically observed within the internal lamina (original magnification, x40). (B) Mean lesion areas of apoE-KO and DKO mice treated with LPS or PBS. Number of mice: apoE-KO: PBS; n= 9, apoE-KO: LPS; n= 9; DKO: PBS; n= 10, DKO: LPS; n= 8. Each symbol represents the mean lesion area of an individual mouse. Horizontal bars represent the mean of all mice in each group, and vertical bars represent SE. Statistical analyses were performed with Mann-Whitney U test. (C) Representative immunohistochemistry of the atherosclerotic lesions from apoE-KO mice administered LPS (right) or PBS (left). Arrows represent atherosclerotic lesions typically observed within the internal lamina (original magnification, × 40 and × 200). Oil red O (ORO) (top), hematoxylin-eosin (HE) (middle) and elastica-Masson staining (EM) (bottom) were performed in each group.

Administration of LPS markedly increased the lesion size in apoE-KO mice compared to the PBS control group (546, 039 ± 81,703 μm2 versus 301,545 ± 43,198 μm2, p = 0.0380) (Fig. 1A, B). By contrast, LPS administration failed to induce alterations in the lesion size in DKO mice (137,083 ± 36,702 versus 149,511 ± 20,113 μm2, p = 0.534). These findings suggest that NKT cells play an important role in the LPS-mediated enhancement of atherosclerotic lesions. The cell-rich area in LPS-treated mice and the collagen-rich area in PBS-treated mice typically appeared prominent (Fig. 1C) when the lesion was stained with elastica-Masson.

Numbers of NKT cells in liver of apoE-KO mice after LPS administration

Next, we analyzed the proportion and absolute number of NKT cells in the liver of each experimental group after LPS or PBS administration. A distinct population of NKT cells (20.74%) was demonstrated as NK1.1+TCRβ+ cells among HMNC of PBS-treated apoE-KO mice (control) (Fig.2A). The proportion (28.11%) and number of NKT cells were significantly increased after LPS administration in apoE-KO mice compared with PBS controls (27.25 ± 0.71 versus 13.63 ± 3.64 × 104, p = 0.0002)(Fig. 2A, B). Few NKT cells were detected in DKO mice treated with either LPS or PBS alone (Fig. 2B).

Figure 2. Flow cytometric analyses of NKT cells in liver of apoE-KO and DKO mice treated with LPS or PBS.

Figure 2

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(A) Representative profiles of flow cytometric analyses of HMNC from apoE-KO and DKO mice treated with LPS or PBS. HMNC from 4 groups of mice were stained with anti-TCRβ and anti-NK1.1 mAb. (B) Mean number of NKT cells in liver of apoE-KO and DKO mice treated with LPS or PBS. Numbers of NKT cells were calculated from flow cytometric data and the actual cell counts. (C) Mean number of iNKT cells or NK1.1+TCRβ+ cells in apoE-KO mice treated with LPS or PBS. Numbers of iNKT cells in liver were calculated from the percentage of α-GalCer/CD1d-tetramer+TCRβ+cells and the actual cell counts. Number of mice is as described in Fig. 1 legend. *p < 0.05

To determine whether the increased population of NKT cells represents invariant NKT (iNKT) cells, which express the Vα14Jα18 TCR and react with α-GalCer, HMNC obtained from LPS- or PBS-treated apoE-KO mice were analyzed with α-GalCer/CD1d-tetramers. Increased numbers of NK1.1+TCRβ+ cells were again clearly demonstrated among HMNC from LPS-treated mice and a similar increased number of α-GalCer/CD1d-tetramer+ cells was observed, as compared with PBS-treated controls (Fig. 2C). In PBS-treated and LPS-treated apoE-KO mice, most of the NK1.1+TCRβ+ cells were positive for α-GalCer/CD1d-tetramer staining (83.46 ± 0.58 versus 83.48 ± 0.79%, respectively), and more than 90% of α-GalCer/CD1d-tetramer positive cells were NK1.1+TCRβ+ cells (83.96 ± 0.85 versus 93.50 ± 1.29%, respectively). These findings demonstrate that most of the expanded NKT cells among the HMNC carry the invariant (Vα14Jα18) TCR.

In the spleen, LPS administration also increased NKT cells and 70~80% of these cellswere iNKT cells (data not shown). Nevertheless, the increase in NKT cells in the spleen was less prominent than that observed in liver (data not shown).

BW and blood chemistry in apoE-KO and DKO mice

Although we administered a low dose of LPS to mice in order to mimic the subclinical level of bacterial infection, systemic influences of endotoxin treatment remained to be clarified. We evaluated the effects of LPS on BW, serum lipid levels and fasting blood sugar (FBS) concentrations 1 wk after the last injection of LPS. No significant difference in the BW was observed between LPS and PBS groups in either apoE-KO, or DKO mice (Fig. 3A). There were also no significant differences in the levels of T-cho, TG, and FBS between the LPS and PBS groups for both apoE-KO and DKO mice (Fig. 3B, D, E). Nevertheless, the HDL-cho levels were slightly decreased in the LPS group compared to the PBS group in both apoE-KO (3.22 ± 0.80 versus 5.78 ± 1.95 mg/dl) and DKO mice (1.63 ± 0.42 versus 7.60 ± 2.85 mg/dl) (Fig. 3C). Of note, a considerable elevation in serum GPT levels was observed in LPS-treated DKO mice compared to the PBS control group (126.5 ± 20.2 versus 37.6 ± 7.3 IU/l, p= 0.0472) (Fig. 3F), whereas no significant difference was noted between LPS-treated apoE-KO mice and PBS controls (49.3 ± 8.7 versus 37.6 ± 7.3 IU/l; p= 0.8275).

Figure 3. BW and blood biochemistry in apoE-KO and DKO micetreated with LPS or PBS.

Figure 3

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Serum samples were obtained from each group of mice 1 week after the last injection of LPS or PBS. Analyses of (A) BW (B) T-chol (C) HDL-chol (D) TG, (E) FBS and (F) GPT were performed. Values are mean ± SE (apoE-KO: PBS; n= 9, apoE-KO: LPS; n= 9; DKO: PBS; n= 10, DKO: LPS; n= 8). *p < 0.05

In vitro and in vivo cytokine production in response to LPS by apoE-KO and DKO mice

Next, we quantified the serum cytokine levels after the last injection of LPS or PBS. In comparison with PBS controls, administration of LPS significantly increased serum levels of TNF-α and IL-2 in both apoE-KO mice (TNF-α 102.9 ± 8.0 versus 61.2 ± 3.4 pg/ml, p= 0.0006; IL-2: 99.0 ± 11.0 versus 57.0 ± 5.5 pg/ml, p= 0.0004) and in DKO mice (TNF-α 99.4 ± 8.9 versus 56.4 ± 10.9 pg/ml, p= 0.0004; IL-2: 95.9 ± 16.6 versus 52.8 ± 3.4 pg/ml, p= 0.0357) (Fig. 4A, B). The levels of IL-4, IFN-γ, and MCP-1 exhibited similar patterns (Fig. 4C, D, E). On the other hand, LPS administration appeared to increase serum IL-12p70 in DKO mice (LPS versus PBS, 142.0 ± 38.3 versus 54.2 ± 20.0, p = 0.1345), whereas LPS treatment decreased the IL-12p70 levels in apoE-KO mice (Fig. 4F). Thus, IL-12p70 levels were significantly higher in LPS-treated DKO mice than in LPS-treated apoE-KO mice (142.0 ± 38.3 versus 22.6 ± 22.6 pg/ml, p = 0.0261). Furthermore, LPS administration significantly or moderately increased serum IL-6 and IL-10 levels, respectively in DKO mice compared to PBS controls (IL-6; 69.4 ± 16.8 versus 16.9 ± 5.8 pg/ml, p = 0.0194; cf. DKO-LPS versus apoE-LPS, 69.4 ± 16.8 versus 24.1 ± 12.4, p = 0.0626) (IL-10; 119.4 ± 63.7 versus 33.5 ± 17.8 pg/ml, p = 0.5368) (Fig. 4G, H).

Figure 4. Ccytokine production in apoE-KO and DKO mice treated with LPS in vivo or in vitro.

Figure 4

Figure 4

Figure 4

Figure 4

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Serum samples were obtained from each group of mice 1 week after the last injection of LPS or PBS. Analyses of (A) TNF-α (B) IL-2 (C) IFN-γ (D) MCP-1 (E) IL-4 (F) IL-12p70 (G) IL-6 and (H) IL-10 were performed with CBA as described in Materials and Methods. Values are mean ± SE (apoE-KO: PBS; n= 9, apoE-KO: LPS; n= 9; DKO: PBS; n= 10, DKO: LPS; n= 8). *p < 0.05 (I) IL-6 and TNF-α production by peritoneal macrophages of apoE-KO or DKO mice elicited with thioglycollate and stimulated with LPS (1 μg/ml, 0.1 μg/ml) in vitro for 24 hr. Cytokines in culture supernatant were quantified as described in Materials and Methods. N.S.: not significant. (J) Effect of co-culture with 2E10 NKT hybridoma cells on production of IL-6 and TNF-α by peritoneal macrophages upon stimulation with LPS. Peritoneal macrophages of apoE-KO or DKO mice were cultured in the presence of LPS (1 μg/ml) with 2E10 hybridoma cells or without hybridoma cells. **p < 0.01; N.S.: not significant. (K) Flow cytometric profiles of intracellular IFN-γ and IL-4 production by hepatic NKT cells 2 hr after α-GalCer, LPS or PBS injection into WT mice. Numbers indicate the percentage of cytokine-positive NKT cells (Δ percentage of isotype control). Representative profile of 3 independent experiments. (L) The intracellular IFN-γ and IL-4 production profile of hepatic NK1.1+TCRβ+ NKT cells in WT mice. Data shown indicate the mean percentage ± SE of cytokine-positive NKT cells from 3 independent experiments.

To examine whether macrophages obtained from DKO mice produced more IL-6 than macrophages from apoE-KO mice, we obtained peritoneal exudate cells (PEC) from either apoE-KO or DKO mice, 4 days after elicitation of macrophages by intraperitoneal injection of 4.05% thioglycollate solution, and stimulated these cells with LPS for 24 hr. We detected similar levels of IL-6 in the culture supernatants from LPS-stimulated PEC derived from apoE-KO and DKO mice (Fig. 4I). Similar to our invivo findings, cultures from apoE-KO and DKO mice produced equivalent levels of TNF-α (Fig. 4I). To examine whether the presence of NKT cells in the cultures affects cytokine production, the elicited PEC cells were co-cultured with NKT hybridoma (clone 2E10) cells in the presence of LPS. IL-6 production appeared to be slightly but significantly suppressed in the presence of 2E10 hybridoma cells for LPS-stimulated PEC derived from both apoE-KO and DKO mice (Fig. 4J). However, no difference was observed for TNF-α production in these cultures.

Although we were unable to identify substantial differences in cytokine production in the serum samples of LPS-treated apoE-KO and DKO mice, we considered the possibility that differnces might only be observed early after LPS stimulation. We therefore isolated spleen cells or HMNC 2 hr after LPS injection, and performed intracellular staining for detection of the cytokines IL-4 and IFN-γ by NKT cells. Indeed, we detected a significant number of iNKT cells that produced IFN-γ in LPS-treated WT mice, although levels were substantially lower compared with α-GalCer treatment (Fig. 4K, L). The percentage of iNKT cells producing IL-4 in LPS-treated mice was minimal as compared with α-GalCer-treated mice.

Effects of LPS on atherosclerosis in apoE-KO mice after depletion of NK cells

We noted that NK cells were also increased in the liver of LPS-treated apoE-KO mice, compared with PBS-treated controls (6.83 ± 0.53 versus 3.46 ± 0.32 × 104, p = 0.0028) (Fig. 5A). On the other hand, no alteration was detected in the numbers of NK cells between the LPS-treated and the control groups in DKO mice. To examine the additional role of NK cells in the exacerbation of atherosclerosis mediated by LPS, we analyzed the lesion areas in apoE-KO mice depleted of NK cells. ApoE-KO mice were administered anti-asialo-GM1 antibody 1 day before LPS treatment. Indeed, LPS administration failed to significantly increase the number of residual NK cells in mice treated with anti-asialo-GM1 (1.911 ± 0.212 versus 1.195 ± 0.134, p = 0.7847) (Fig. 5B). These NK cell-depleted apoE-KO mice demonstrated no significant aggravation in lesion size upon LPS administration (287,110 ± 75,803 versus 202,594 ± 42,633 μm2, p = 0.6015) (Fig. 5C).

Figure 5. Flow cytometric analysis of liver NK cells and atherosclerotic lesion areas in apoE-KO mice depleted of NK cells and treated with LPS.

Figure 5

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(A) Numbers of NK cells in liver from apoE-KO or DKO treated with LPS or PBS were calculated from flow cytometric data and the actual cell counts. (B) Mean number of NK cells in liver from apoE-KO mice depleted of NK cells with rabbit anti-asialoGM1 Ab as described in Materials and Methods. Numbers of NK cells in liver were calculated from flow cytometric data and the actual cell counts (NK depletion: PBS; n= 5, NK depletion: LPS; n= 5). (C) Mean lesion areas of apoE-KO mice depleted of NK cells (NK depletion) and administered LPS or PBS. Each symbol represents the lesion area of an individual mouse. Horizontal bars represent the mean of all mice in each group, and vertical bars represent SE. * p < 0.05

Discussion

It has been reported that LPS-induced exacerbation of atherosclerosis is associated with an increase of NKT cells in various organs, including atherosclerotic lesions (Ostos et al. 2002). Although NKT cells were shown to accelerate the development of atherosclerosis in mouse models (Tupin et al. 2004; Nakai et al. 2004; Major et al. 2004; Aslanian et al. 2005), the involvement of NKT cells in the exacerbation of atherosclerosis in the presence of bacterial products has not been directly investigated. In the present study, using apoE-KO mice and apoE-KO/CD1d-KO (DKO) mice that lack CD1d-restricted NKT cells, we demonstrated that NKT cells were indeed responsible for the LPS-mediated exacerbation of atherosclerosis. Compared to PBS-treated controls, the number of NKT cells was increased and the development of atherosclerotic lesions was accelerated in LPS-treated apoE-KO mice but not in LPS-treated DKO mice. We have previously reported that activation of NKT cells by chronic α-GalCer stimulation alters the quality of atherosclerotic lesions from collagen rich to high cellularity (Nakai et al. 2004). Our present results similarly demonstrated that activation of NKT cells tended to induce high cellularity in the lesions (Fig. 1C).

NKT cells have been shown to directly recognize microbial lipids such as α-galacturonylceramide (GSL-1) from Sphingomonas spp and Ehrlichia muris (Kinjo et al. 2005; Mattner et al. 2005) and α-galactosyldiacylglycerol (MGDG) from Borrelia burgdorferi (Kinjo et al. 2006) in the context of CD1d as represented by α-GalCer administration (Nakai et al. 2004) (direct mechanism of NKT cell activation shown in Fig. 6). It has been reported that NKT cells can also be activated during Salmonella typhimurium infection, in an indirect mechanism that may involve endogenous NKT cell ligands, for example, isoglobotriaosylceramide (iGb3) (Mattner et al. 2005; De Libero et al. 2005) (Fig. 6). In the latter, indirect mechanism of NKT cell activation, LPS may induce the production of cytokines that synergize with endogenous ligands for activation of NKT cells (Brigl et al. 2003). These direct and indirect mechanisms of NKT cell activation are now considered as distinct recognition modes by which NKT cells can be activated by a wide variety of microbes (Kronenberg and Kinjo 2009; Matsuda et al. 2008). Recent studies have provided significant insight into the molecular basis for indirect activation of NKT cells by LPS. One research group showed that LPS inhibits α-galactosidase A, an enzyme that degrades iGb3 and thus activates NKT cells by elevating iGb3 levels in vivo in an MyD88-dependent fashion (Darmoise et al. 2010). Another group demonstrated that TLR signaling activates β-GlcCer synthetase and simultaneously inhibits lactosylceramide synthetase, thus elevating expression of an endogenous ligand, β-GlcCer, resulting in NKT cell activation (Brennan et al. 2011). These or similar pathways may be involved in various situation of NKT cell activation. According to this model, it is likely that repeated LPS administration to apoE-KO mice, employed in the present study, chronically activates NKT cells through the indirect pathway. Indeed, LPS administration to apoE-KO mice not only aggravated atherosclerosis but also increased the number of NKT cells in the liver and spleen of these mice.

Fig. 6. Schematic representation of the relationship between exacerbation of atherosclerotic lesions by microbial lipids and activation of NKT cells by microbial lipids.

Fig. 6

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NKT cells may aggravate the lesion development of atherosclerosis via soluble or cellular factors produced in response to direct activation by pathogen-derived glycolipids (represented by α-GalCer) or indirect activation by LPS via recognition of self-derived glycolipids in the presence of CD1d.

We showed that the number of NK cells was also increased in the liver of LPS-treated apoE-KO mice (Fig. 5). Since no change was noted in the NK cell number in HMNC between LPS-treated and PBS-treated DKO mice (Fig. 5), we considered that the activation of NKT cells was essential for the increase of NK cells in the LPS-treated apoE-KO mice. It has been reported that NKT cell activation leads to IFN-γ production, which subsequently results in NK cell activation (Kitamura et al. 1999) and propagates the initial effects. Thus, NK cell depletion may result in diminution of pro-atherogenic influences induced following NKT cell stimulation. To directly elucidate NK cell functions in atherosclerosis development, we analyzed NK cell-depleted apoE-KO mice. The number of NK cells was substantially and moderately reduced in HMNC and splenocytes, respectively, following anti-asialo-GM1 treatment (compare Fig. 2 with Fig. 5 for HMNC; data not shown for spleen). Our results showed that LPS administration failed to enhance the atherosclerotic lesions size in NK cell-depleted apoE-KO mice (Fig. 5). Consistent with these findings, Whitman et al reported that the transfer of bone marrow cells from NK cell-deficient, Ly49A transgenic (Tg) mice induced lethally irradiated LDLR-KO recipients to develop smaller atherosclerotic lesions compared with LDLR-KO recipients transferred with wild-type bone marrow cells (Whitman et al. 2004). Thus, our findings indicate that activation of NKT cells by LPS leads to activation of NK cells, and the activated NK cells further promote lesion development. Synergistic effects between NKT and NK cells have been reported also in tumor immunity (Smyth et al. 2005).

Concerning the serum cytokine levels, a significant elevation of TNF-α and IL-2, and a moderate increase of IFN-γ, IL-4, and MCP-1 levels were induced by LPS administration in both apoE-KO and DKO mice (Fig. 4), suggesting that NKT cells are dispensable for the elevation of these cytokines. On the other hand, the absence of NKT cells was associated with significant augmentation of LPS-induced IL-12p70 and IL-6 production, and a slight increase in IL-10. It should be noted that isolated peritoneal macrophages from apoE and DKO mice produced similar amounts of IL-6 and TNF-α in vitro upon stimulation with LPS, suggesting that CD1d disruption in the apoE-deficient background did not cause macrophages to produce increased levels of IL-6. However, addition of 2E10 NKT hybridoma cells lowered the level of IL-6 but not TNF-α in these cultures, but this was independent of CD1d expression by the macrophages (Fig. 4J). Moreover, this effect was only observed for IL-6 production. Of note, IL-10 and IL-12, which tended to be increased in the serum of DKO mice, could not be detected in these in vitro cultures. The mechanism for this differential cytokine induction is unknown and might be due to suppression of cytokine production, cytokine absorption by cells, or other mechanisms, which will be pursued in future studies. Of note, a significant elevation of GPT, a marker of liver injury, was seen in LPS-treated DKO mice that showed elevations in IL-12p70 but not in LPS-treated apoE-KO mice (Fig. 4). These findings are consistent with a report that IL-12 and IL-18 produced by HMNC play important roles in LPS-induced liver injury (Ono et al. 2003). However, the role of the increased levels of IL-6 and IL-10, both of which have been reported as protective cytokines against LPS-induced liver injury (Inoue et al. 2005; Louis et al. 1997), in the LPS-treated DKO mice is hard to interpret at the present time. The relevance of this contradictory pattern in cytokine production will be investigated in future studies. Nevertheless, with regard to the lesion development of atherosclerosis induced by LPS administration, the presence and the activation of NKT cells seem to play more important roles than the systemically increased levels of pro-atherogenic cytokines.

TLR-KO and MyD88 adaptor-KO mice have already been examined for the lesion enhancement of atherosclerosis (Erridge 2009). Indeed, the disruption of TLR4 that is critical for the recognition of LPS (Yamamoto et al. 2009) in apoE-KO (apoE/TLR4-DKO) mice reduced the size and altered the phenotype of plaques with less macrophage infiltration and inflammatory changes. These findings are consistent with the results of our studies and might be due to defective activation of NKT cells via the indirect pathway. It should also be noted that the lesion development in apoE/TLR4-DKO was significantly reduced in comparison with that of apoE-KO mice, even without the administration of LPS (Michelson et al. 2004). This enhancement of the atherosclerotic lesion by the presence of TLR4 is likely due to the recognition of low level of oral cavity-derived (Maekawa et al. 2011) and/or colon-derived LPS (Lindros et al. 2005), and/or the natural ligands such as oxidized lipids, free fatty acids, and other modified lipids derived from food or the environment (Björkbacka 2006). TLR4 is expressed on macrophages in murine and human lipid-rich atherosclerotic plaques (Xu et al. 2001) and was up-regulated by oxidized LDL, which might result in enhanced activation of macrophages in situ. We have previously shown that CD1d molecules are also up-regulated on macrophages following stimulation with oxidized LDL (Nakai et al. 2004). Thus, increased TLR4 and CD1d expression may also contribute to the enhanced NKT cell activation and the exacerbation of atherosclerotic lesions in LPS-treated apoE-KO mice.

In the present study, we have compared the lesion size between apoE-KO and DKO mice. The latter animals lack both type I NKT cells (also called iNKT cells) that express an invariant TCR and react with α-GalCer, and type II NKT cells, wich express more diverse receptors and lack α-GalCer-reactivity but share with type I NKT cells the capacity to produce a variety of immunoregulatory cytokines (Godfrey et al. 2007). Since the effects of LPS on NKT cells appeared to be mainly mediated through activation of type I NKT cells (Darmoise et al. 2010; Brennan et al. 2011), the effects of CD1d-deficiency on LPS-exacerbated atherosclerosis are most likely due to the absence of type I NKT cells. Nevertheless, it remains possible that type II NKT cells contribute as well. A previous study reported 30% reduction in lesion size in LDLR/Jα18 DKO (Jα-DKO) mice compared with LDLR-KO mice, suggesting that the remaining, type II NKT cells that are present in Jα18-KO mice promote atherosclerosis (Rogers et al. 2008). Similarly, we also observed that, in mice fed an atherogenic diet, Jα18-KO mice exhibited smaller atherosclerotic lesion than WT mice, but larger than CD1d-KO mice (Nakai et al. 2004; unpublished observations).

In conclusion, NKT cells play a critical role in LPS-enhanced atherosclerosis. NKT cells may be activated not only by lipid stress but also by constant exposure to pathogens or pathogen-derived components in the presence of activated macrophages. The present study demonstrates the importance of controlling NKT cell activation by reducing both hyperlipidemic stress and bacterial products for enduring treatment of atherosclerosis.

Acknowledgments

We would like to thank Kirin Brewery Company for providing α-GalCer. The present study was supported by Grants-in-Aid for Scientific Research B (#20390106, #23370059), and C from the Japan Society for the Promotion of Science (JSPS) (K.I., N.I., S.F.), by Global COE Program, Establishment of International Collaboration Center for Zoonosis Control, from the Ministry of Education, Culture, Science, Sports and Technology (MEXT), Japan (K.I.), by a Health and Labor Sciences Research Grant on Intractable Diseases from the Ministry of Health, Labor and Welfare of Japan (K.I.), grants from the Akiyama Foundation (K.I, Y.N., T.M), Daiwa Securities Health Foundation (K.I., Y.N., S.F.), The Suhara Memorial Foundation (K.I., Y.N., S.F.), BioLegend/Tomy Digital Biology (M. S.), and Parent’s Association (Keyakikai) at Kitasato University School of Medicine (M. S.); and by National Institutes of Health grants AI070305, HL089667 and DK081536 (L.V.K.).

Footnotes

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