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The Journal of Immunology, 2006, 177: 26-30.
Copyright © 2006 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: Impaired Glycosphingolipid Trafficking and NKT Cell Development in Mice Lacking Niemann-Pick Type C1 Protein1

Yuval Sagiv*, Kelly Hudspeth*, Jochen Mattner*, Nicolas Schrantz{dagger}, Randi K. Stern*, Dapeng Zhou{ddagger}, Paul B. Savage§, Luc Teyton{dagger} and Albert Bendelac2,*

* Committee on Immunology, University of Chicago, Chicago, IL 60637; {dagger} Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037; {ddagger} MD Anderson Cancer Center, University of Texas, Houston, TX 77030; and § Department of Chemistry, Brigham Young University, Provo UT 84602


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Niemann-Pick Type C1 (NPC1) is a late endosomal/lysosomal transmembrane protein involved in the cellular transport of glycosphingolipids and cholesterol that is mutated in a majority of patients with Niemann-Pick C neurodegenerative disease. We found that NPC1-deficient mice lacked V{alpha}14-J{alpha}18 NKT cells, a major population of CD1d-restricted T cells that is conserved in humans. NPC1-deficient mice also exhibited marked defects in the presentation of Sphingomonas cell wall Ags to NKT cells and in bacterial clearance in vivo. A synthetic fluorescent {alpha}-glycosylceramide analog of the Sphingomonas Ag trafficked to the lysosome of wild-type cells but accumulated in the late endosome of NPC1-deficient cells. These findings reveal a blockade of lipid trafficking between endosome and lysosome as a consequence of NPC1 deficiency and suggest a common mechanism for the defects in lipid presentation and development of V{alpha}14-J{alpha}18 NKT cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Niemann-Pick Type C1 (NPC1)3 is a large endosomal/lysosomal glycoprotein conserved from yeast to mammals with 13 predicted membrane-spanning domains that include a potential sterol-sensing domain (1, 2). Mutations in the NPC-1 gene are found in 95% of patients with NPC disease, a neurodegenerative disorder associated with lipid storage (3). NPC mutant cells characteristically exhibit an accumulation of cholesterol and glycosphingolipids (GSLs) in organelles resembling late endosomes (LEs) (4, 5, 6). However, the precise function of NPC1 remains elusive, and it is unclear at present which of these defects is a direct or indirect consequence of NPC1 deficiency in vivo (7, 8, 9, 10).

Lipid transport is essential to the processing and presentation of lipid Ags to CD1-restricted T lymphocytes. The CD1 family of glycoproteins is composed of MHC-like, beta2-microglobulin-associated glycoproteins that specialize in the capture and presentation of self and microbial glycolipid and lipopeptide Ags to T cells (11, 12). Several lipid transfer proteins (LTPs) have been shown recently to be involved in CD1-mediated lipid Ags presentation. Saposins, a family of LTPs involved in lysosomal GSL degradation (13), also perform essential lipid exchange reactions between membranes and CD1 proteins (14, 15, 16). In the mouse system, where CD1d is the only CD1 family member, prosaposin-deficient mice lacked V{alpha}14-J{alpha}18 NKT cells, a specialized subset of T cells with dual specificity for the self GSL isoglobotrihexosylceramide (iGb3) (17) and for microbial {alpha}-glycuronosylceramides (18, 19, 20). Microsomal triglyceride transfer protein was recently suggested to function in the CD1 pathway as well, based on alterations of CD1d trafficking and NKT cell function in conditional mutant mice (21, 22). Thus, the uptake and trafficking of lipids and their processing and loading onto CD1 molecule may rely on a set of preexisting proteins already involved in general lipid metabolism. In this study, we have examined CD1-mediated lipid presentation in mutant mice and cells lacking NPC1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Disclosures
 References
 
Mice

BALB/cNctr-Npc1m1N/J mice were obtained from The Jackson Laboratory. Littermates obtained from heterozygous matings were genotyped by PCR and used for comparative analysis. CD1d–/– mice were in the C57BL/6 background (23). All mice were raised in a specific pathogen-free environment at the University of Chicago, according to the Institutional Animal Care and Use Committee guidelines.

Cell culture

Fresh thymocytes, splenocytes, and cultured bone marrow-derived dendritic cells (BMDCs) were obtained as described (24). Parental JP17 and NPC1-mutant A101 Chinese hamster ovary (CHO) cells were a gift from K. Higaki (Faculty of Medicine, Tottori University, Yonago, Japan) (25).

Flow cytometry

CD1d-{alpha}-galactosylceramide ({alpha}-GC) tetramers were generated and used as described (26). PE-conjugated anti-CD1d Abs were obtained from BD Pharmingen. Flow cytometry was performed as described with FACSCalibur (BD Biosciences), and data were analyzed using CellQuest Pro software (BD Biosciences).

T cell hybridoma stimulation assay

NKT hybridomas included the V{alpha}14-J{alpha}18 DN32.D3 and the non-V{alpha}14 TCB11 and TBD7 clones. Stimulation of 5 x 104 hybridoma cells with fresh thymocytes (5 x 105), splenocytes (5 x 105), or with BMDCs (5 x 104) was as described (17). CHO cells were used at 5 x 104 per well. IL-2 released in cultured supernatants was measured using CTLL-2 indicator cells as described (27).

Bacterial stimulation assay

Sphingomonas paucimobilis (ATCC 29837) was grown in Mueller-Hinton Agar as described (19). Heat kill of bacteria was by exposure to 74°C for 2 h. A total of 5 x 105–5 x106 CFU equivalent were added per well containing BMDCs (5 x 104) for stimulation of the DN32.D3 hybridoma.

Live infection

S. paucimobilis was grown for 8 h at 37°C to an OD600 of 0.5, washed, and diluted in PBS. NPC1+/+ and NPC1–/– littermates were injected i.v. with 1 x 107 bacteria in 100 µl. Twenty-four hours after infection, bacterial counts were performed after tissue homogenization of liver and spleen in 0.5% Triton X-100 and serial dilution of the homogenate in Mueller-Hinton broth before culture.

Confocal microscopy

To study lipid trafficking, cells were incubated with 5 µM N-(5-(5,7-dimethylborondipyrromethenediflouride)-1-pentanoyl)D-lactosphingosine (BODIPY)-LacCer (Molecular Probes) or with 9 µM prodan-conjugated {alpha}-GC (PBS10) (28) at 37°C, for 1 h or overnight, respectively. Cells were directly analyzed by confocal microscopy or fixed before staining with Abs as described (14). When cells were fixed, slides were examined by confocal microscopy without delay to minimize the leakage of lipids out of their original compartment. LysoTracker Red DND-99 (Molecular Probes) was titrated to 1/104 dilution for 5 min on DCs, and 1/4 x 103 dilution for 30 min on CHO cells to stain lysosomes exclusively. The following Abs were used: anti-M6PR IgY Abs, Cy5-conjugated goat anti-chicken IgY (Abcam); Armenian hamster anti-mouse saposin B and saposin C (used as a mixture; N.S. and L.T., manuscript in preparation); and FITC-conjugated goat anti-Armenian hamster IgG (Jackson ImmunoResearch Laboratories). Cells were examined by confocal microscopy using a confocal microscope (TCS SP2 AOBS; Leica), with a 63X NA1.4 oil objective lens, at room temperature.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Disclosures
 References
 
NPC1–/– mice exhibited a massive loss of V{alpha}14-J{alpha}18 NKT cells, compared with NPC+/+ littermates. This NKT cells deficiency was comparable to that of CD1d–/– mice, both in the thymus and the spleen, as judged by specific staining with CD1d tetramers loaded with the synthetic agonist ligand of NKT cells, {alpha}-GC (Fig. 1A). The NKT cell defect was specific as judged by the normal composition of the CD4, CD8, and B cell compartments (data not shown). The stimulation of the V{alpha}14-J{alpha}18 NKT hybridoma DN32.D3 by natural lipid ligands on the surface of thymocytes, such as CD1d–iGb3 complexes (17), was impaired (Fig. 1B). In contrast, the stimulation of control, non-V{alpha}14 hybridomas such as TCB11 and TBD7, which recognize unidentified ligands loaded outside of the endosomal/lysosomal compartment (23), was preserved (Fig. 1B), as was CD1d expression in relevant cell types such as thymocytes, splenocytes, and DCs (Fig. 1C).


Figure 1
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  		FIGURE 1. Impaired selection of V{alpha}14 NKT cells in NPC1-deficient mice. A, Upper, V{alpha}14 NKT cells in the thymus and spleen of NPC1+/+ and NPC1–/– littermates were stained by CD1d-{alpha}-GC tetramers and compared with age-matched CD1d–/– mice. Percentages of cells in the V{alpha}14 NKT cell gate are indicated. Lower, Percentages of V{alpha}14 NKT cells in NPC1–/– mice, compared with NPC1+/+ littermates after subtraction of background (CD1d–/– mice). A summary of three independent experiments is shown. B, IL-2 response of the V{alpha}14 NKT hybridoma DN32.D3 and the non-V{alpha}14 hybridomas TCB11 and TBD7 stimulated by fresh thymocytes. A summary of three independent experiments is shown. C, Surface expression of CD1d in thymocytes, splenocytes and BMDCs from +/+ (thin lines), –/– (thick lines), and control CD1d KO (filled histogram) mice. D, Presentation of {alpha}-GC and Gal {alpha}1,2 Gal {alpha}1,1 Cer by BMDCs from NPC1+/+ (•), NPC1–/– ({circ}), and CD1 KO ({triangleup}) mice, as measured by IL-2 release from the DN32.D3 hybridoma.

 
The lysosomal compartment is particularly important for the generation of natural NKT ligands and their loading onto CD1d. For example, iGb3 is generated from the degradation of iGb4 in the lysosome and loaded onto CD1d with the assistance of LTPs such as saposins (14). We thus tested the possibility that the NKT cell phenotype may be related to defective lysosomal transport of GSLs. Consistent with this hypothesis, presentation of exogenous GSL Ags by DCs (Fig. 1D) or whole spleen (data not shown) was altered. Thus, presentation of Gal{alpha}1,2 Gal {alpha}Cer, a diglycosylated variant of {alpha}-GC that requires lysosomal processing into {alpha}GC before recognition by NKT cells, was massively impaired. Presentation of {alpha}-GC was less impaired, likely because it can also load CD1d outside of the lysosomal compartment. In these experiments, due to low levels of CD1d expression by DN32.D3, some residual stimulation was observed even in the presence of CD1d-deficient APCs.

In vivo clearance of Sphingomonas, a Gram-negative bacterium expressing {alpha}GC NKT ligands instead of LPS in its cell wall, was profoundly impaired (Fig. 2A), consistent with the role of V{alpha}14-J{alpha}18 NKT cells in detecting and rejecting this class of bacteria (19, 20). Upon in vitro exposure to heat-killed or live Sphingomonas, NPC1–/– BMDCs failed to activate the V{alpha}14-J{alpha}18 NKT hybridoma DN32.D3 (Fig. 2B), further suggesting the presence of defects in lipid presentation to the immune system.


Figure 2
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  		FIGURE 2. Susceptibility of NPC1-deficient mice to Sphingomonas infection. A, NPC1–/– and WT littermates mice were injected with 1 x 107 bacteria i.v., and the number of bacteria (CFU) in liver and spleen was measured 24 h later by limiting dilution assay. B, Presentation of heat-killed (H.K.) or live Sphingomonas at the indicated ratio of bacteria to BMDCs, as measured by IL-2 release from the DN32.D3 hybridoma.

 
To directly visualize lipid transport, we used confocal microscopy to examine the intracellular trafficking of a fluorescently labeled GSL ligand of NKT cells. Fluorescent GSL are useful tools for the study of GSL sorting and trafficking in normal and mutant cells (29, 30). Following internalization from the plasma membrane, some fluorescent GSLs were reported to recycle between the plasma membrane and intracellular membranes (recycling endosomes) (31, 32, 33), while others were delivered to the LE and further transferred to the Golgi complex (30, 34). However, none of these fluorescent lipids were found to access the lysosome where GSLs are known to undergo degradation and loading onto CD1d. Lactosylceramide (Gal beta1,4 Glc beta1,1 Ceramide) with a short acyl chain tagged with BODIPY (BODIPY-LacCer) (34), which normally accumulates in the Golgi of WT cells, was broadly used in recent years to demonstrate retention of GSLs in endosomal compartments of NPC1-deficient CHO cells and human fibroblasts (9, 35, 36). Consistent with these reports, we observed that exogenously administered BODIPY-LacCer rapidly reached the Golgi in BMDCs derived from wild-type mice but markedly accumulated in endosomal compartments of NPC1-deficient cells (data not shown).

We recently reported the synthesis of PBS10, an {alpha}-GC mimic of the microbial NKT ligands, where we appended the fluorophore prodan to the carbohydrate in C6" (Fig. 3A). PBS10 binds CD1d and is recognized by NKT cells in a manner similar to the unaltered microbial ligands themselves, or to the synthetic {alpha}GC (28). In wild-type DCs, PBS10 clearly accumulated in the lysosomal compartment as shown by the extensive colocalization with the lysosomal marker LysoTracker Red (Fig. 3A). In these experiments, LysoTracker Red was used under conditions that stained the lysosome and excluded the LE. Lysosomal accumulation was further confirmed by colocalization with LAMP-1, but not with the LE marker mannose 6 phosphate receptor (M6PR) (data not shown). Thus, in contrast with BODIPY-LacCer, PBS10 provided a unique tool to examine lipid transport from LE to lysosome, which is considered to be the relevant pathway for the presentation of endogenous and bacterial ligands to V{alpha}14 NKT cells (14, 15, 16, 17, 37, 38, 39). Remarkably, PBS10 consistently failed to reach the lysosome of NPC1-deficient cell over a time course of up to 24 h (Fig. 3A). Instead, PBS10 accumulated in the M6PR+ LE compartment (Fig. 4B and data not shown), indicating that the NPC1 deficiency is associated with a more general blockade of lipid exit out of the LE than previously appreciated.


Figure 3
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  		FIGURE 3. Impaired trafficking of GSLs in NPC1-deficient BMDCs. A, BMDCs derived from NPC1+/+ and NPC1–/– littermates were incubated with prodan-conjugated {alpha}-GC (PBS10, green), followed by staining with LysoTracker Red as a lysosomal marker (red) before confocal microscopy. (Scale bar, 8 µm.). The structure of PBS10 is depicted on the right side. B, BMDCs were incubated with PBS10 (blue), anti-CD1d Abs (red), and anti-saposin B and C Abs (green).

 

Figure 4
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  		FIGURE 4. Modest restoration of GSLs trafficking by NB-DGJ. A, Upper panels, JP17 and A101 cells were incubated with prodan-conjugated {alpha}-GC (PBS10, green) overnight followed by staining with LysoTracker Red. (Scale bar, 8 µm). Lower panels, A101 cells were incubated with the indicated amounts of NB-DGJ (µM) for 48 h, before staining as above; B, JP17 and A101 cells were pulsed with PBS10 (blue) and BODIPY-LacCer (green) simultaneously, then fixed and stained with anti-M6PR (red). (Scale bar, 10 µm.).

 
To further examine the respective cellular distribution of proteins involved in CD1d-mediated lipid presentation, we performed double staining for CD1d and saposin after pulse with the fluorescent GSLs. In wild-type DCs, there was clear colocalization, (white), of PBS10 with CD1d and saposins in the lysosomal compartment (Fig. 3B). Saposins or their precursor prosaposin (green) also were found in other subcellular compartments, such as LE and the Golgi (Fig. 3B and data not shown). In NPC1–/– DCs, however, PBS10 was absent from the lysosomes where most of the CD1d molecules were still found, whereas saposins were found both in the lysosome with CD1d (yellow) and in the LEs with PBS10 (light blue) (Fig. 3B). These findings suggest therefore that, by selectively preventing the transport of PBS10 from LEs to lysosome, the NPC1 mutation impairs access to the most abundant pool of CD1d in the low pH environment needed for the maturation and function of saposins.

The precise function of NPC1 in lipid transport remains uncharacterized. Interestingly, however, it has been suggested that lipid accumulation per se, independently of the NPC1 mutation, could constitute a self-aggravating disorder (40). To test this possibility, we used N-butyldeoxygalactonojirimycin (NB-DGJ), a compound that ameliorates lipid storage disease by inhibiting the synthesis of GSLs, to test this possibility. NB-DGJ was shown previously to restore the Golgi transport of BODIPY-LacCer accumulated in endosomal compartment of NPC1-deficient cells (40, 41). Because NB-DGJ exhibited toxicity against BMDCs at pharmacological doses (data not shown), we used the CHO cell line A101 carrying the NPC1 gene inactivated by retroviral gene trap mutagenesis and the parent line JP17 as a model (25). As expected, PBS10 accumulated in the lysosomal compartment of wild-type JP17 cells, as shown by colocalization with LysoTracker Red, but failed to reach the lysosome in NPC1-deficient A101 cells (Fig. 4A, upper panels). Instead, like BODIPY-LacCer, PBS10 accumulated in the M6PR+ LE compartment of A101 cells (Fig. 4B). Interestingly, after treatment with NB-DGJ for 48 h, we could detect a modest but consistent restoration of lysosomal transport of PBS10 (yellow) in A101 cells at the highest doses of inhibitor (Fig. 4A, lower panels). This finding suggests that the lack of NPC1 does not completely abrogate GSL transport to the lysosome and supports the notion that GSLs and cholesterol accumulation in NPC1-deficient cells also contribute to some degree to the blockade of the NKT ligand PBS10 in the LE.

Altogether, these results demonstrate a developmental loss of NKT cells in mice lacking the lipid transporter NPC1 and identify a block in GSL transport from LE to lysosome as a previously undocumented effect of the mutation and a potential mechanism underlying defects in natural and bacterial GSL ligand presentation by CD1d. Future studies in human are warranted to determine whether patients harboring NPC1 mutations also exhibit such unexpected immunological phenotype.


   Acknowledgments
 
We thank the Bendelac laboratory members for expert advice and discussion, and Dr. Vytas Bindokas for suggestions and help with confocal microscopy.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI38339 (to A.B.) and PO1AI053725 (to A.B., P.B.S., and L.T.), and Cancer Research Institute Fellowships (to Y.S. and D.Z.). A.B. is a Howard Hughes Medical Institute Investigator. Back

2 Address correspondence and reprint requests to Dr. Albert Bendelac, Department of Pathology, AMB P309, MC1089, University of Chicago, Chicago, IL 60637. E-mail address: abendela{at}bsd.uchicago.edu Back

3 Abbreviations used in this paper: NPC1, Niemann-Pick Type C1; GSL, glycosphingolipid; LE, late endosome; LTP, lipid transfer protein; iGb3, isoglobotrihexosylceramide; BMDC, bone marrow-derived dendritic cell; CHO, Chinese hamster ovary; {alpha}-GC, {alpha}-galactosylceramide; NB-DGJ, N-butyldeoxygalactonojirimycin; BODIPY-LacCer, N-(5-(5,7-dimethylboron-dipyrromethenediflouride)-1-pentanoyl)D-lactosphingosine. Back

Received for publication April 3, 2006. Accepted for publication May 9, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 

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