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The Journal of Immunology, 2008, 180, 4425 -4432
Copyright © 2008 by The American Association of Immunologists, Inc.
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Inhibition of TLR Activation and Up-Regulation of IL-1R-Associated Kinase-M Expression by Exogenous Gangliosides1

Weiping Shen2,*,{dagger}, Kelly Stone2,*,{dagger}, Alessandra Jales*, David Leitenberg{ddagger} and Stephan Ladisch3,*,{dagger},§

* Center for Cancer and Immunology Research, Children’s Research Institute, Children’s National Medical Center, and {dagger} Department of Pediatrics, {ddagger} Department of Microbiology, Immunology, and Tropical Medicine, and § Department of Biochemistry and Molecular Biology, George Washington University School of Medicine, Washington, DC


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Gangliosides, sialic acid-containing glycosphingolipids present in the outer leaflet of plasma membranes, are produced at high levels by some tumors, are actively shed into the tumor microenvironment, and can be detected in high concentrations in the serum of cancer patients. These tumor-shed molecules are known to be immunosuppressive, although mechanisms remain to be fully elucidated. In this study, we show that membrane enrichment of human monocytes with purified exogenous gangliosides potently inhibits ligand-induced activation and proinflammatory cytokine production induced by a broad range of TLRs, including TLR2, TLR3, TLR6, and TLR7/8, in addition to a previously identified inhibitory effect on TLR4 and TLR5. Inhibition of TLR activation is reversible, with complete restoration of TLR signaling within 6–24 h of washout of exogenous gangliosides, and is selective for certain gangliosides (GM1, GD1a, and GD1b), whereas others (GM3) are inactive. To characterize the inhibition, we assessed the expression of the TLR signaling pathway inhibitor, IL-1 receptor associated kinase-M (IRAK-M). In response to ganglioside enrichment alone, we observed striking up-regulation of IRAK-M in monocytes, but without concomitant proinflammatory cytokine production. This contrasts with endotoxin tolerance, in which IRAK-M up-regulation follows proinflammatory cytokine expression caused by LPS exposure. We hypothesize that ganglioside treatment induces a state of tolerance to TLR signaling, leading to blunted activation of innate immune responses. In the tumor microenvironment, shed tumor ganglioside enrichment of APC membranes may likewise cause these cells to bypass the normal TLR signaling response and progress directly to the inhibitory state.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Gangliosides are sialic acid-containing membrane glycosphingolipids that are present predominantly in the outer leaflet of cell membranes. As tumor cells proliferate, gangliosides are shed into their local microenvironment both in vitro and in vivo (1, 2, 3, 4), particularly by tumors of neuroectodermal origin. Many different molecular species are shed, and generally they reflect the ganglioside complement of the tumor cells. Shed tumor gangliosides have been detected in the circulation of some patients, and in neuroblastoma, for example, serve both qualitatively as tumor markers (5) and quantitatively as prognostic indicators of subsequent outcome (6).

Shed gangliosides are immunosuppressive and likely play a role in evasion of tumor destruction by local immune cells, as demonstrated in several murine models (1, 7, 8, 9). Multiple immunosuppressive activities have been ascribed to gangliosides, but molecular mechanisms (10) remain to be fully elucidated. Since gangliosides are amphiphatic molecules capable of binding to membranes, some hypotheses to explain these effects include alteration of membrane protein conformations or disruption of cell membrane organization and dynamics (11).

Exogenous gangliosides inhibit Ag processing and presentation (12, 13), lymphocyte proliferation (14, 15, 16), Th cell differentiation (17), and activation of mast cells (18). We and others have previously reported that pretreatment with gangliosides inhibits differentiation and activation of APCs (dendritic cells (DC)4 and monocytes) following stimulation with LPS, including failure to up-regulate expression of proinflammatory cytokines and cell surface costimulatory molecules CD80 and CD86 (12, 19, 20, 21).

Since both LPS-induced activation of DC and monocytes (19, 22) and flagellin-induced TLR5 signaling (23) are inhibited by exogenous gangliosides, we reasoned that a defect in the TLR signaling pathway might be an important mechanism for ganglioside-induced immunosuppression. TLRs are type I membrane-spanning receptors that are expressed on APCs, among other types of cells. The essential role of TLRs is to recognize pathogen-associated molecular patterns (24), including LPS from Gram-negative bacteria, lipoteichoic acid (LTA) from Gram-positive bacteria, and double- or single-stranded RNA from viruses, and to serve as a mechanism for initiating "danger signals" to activate the immune system (25, 26, 27, 28). As such, TLRs are an integral part of the innate immune system. Binding of pattern-containing molecules from microorganisms or host molecules to TLRs leads to formation of TLR homodimers, heterodimers, or larger aggregates, and recruitment to lipid rafts (29) with subsequent recruitment of adaptor molecules, such as MyD88, Toll-IL-1R domain-containing adaptor protein, Toll-IL-1R domain-containing adaptor-inducing IFN-β, and/or Toll-IL-1R domain-containing adaptor-inducing IFN-related adaptor molecule. Association of the adaptor molecules with the receptors leads to activation of the signaling pathway, with recruitment and activation by phosphorylation of IRAK-1, IRAK-4, and TRAF6, and eventual translocation of NF-{kappa}B to the cell nucleus (30). The end result of TLR stimulation is up-regulation of proinflammatory cytokine gene expression, including IL-6, IL-12, and TNF-{alpha}, and up-regulation of costimulatory molecules, including CD80 and CD86 (31, 32, 33).

Negative regulators of TLR signaling have also been described, including the receptors single immunoglobulin IL-1R-related protein and ST2, the inactive kinase IRAK-M, SOCS1, and the E3 ubiquitin-protein ligase, Triad3A (34, 35, 36, 37, 38, 39). These negative regulators are important for down-modulating TLR responses, and disruption of these mechanisms leads to exaggerated inflammatory responses.

In this study, we characterized the inhibitory effect of exogenous gangliosides on TLR signaling, using a range of TLR ligands to activate the APC. Our findings demonstrate a broad effect of gangliosides on TLR activation, by multiple different ligands, and they identify a previously unknown mechanism by which this may occur, direct induction of the TLR pathway inhibitor IRAK-M.


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

Highly purified gangliosides (GD1a, GD1b, GM1, and GM3) were purchased from Matreya and stored at –20°C. Before use, the gangliosides [free of endotoxin, as assessed by the Limulus assay (Cambrex)] were dissolved in serum-free RPMI 1640 medium and resuspended by mild-bath sonication. LTA (TLR 2 ligand), Poly (cytidylic-inosinic) acid (poly(I:C)) (TLR 3 ligand), and LPS (TLR 4 ligand) were purchased from Sigma-Aldrich. Zymosan A (TLR 2 and 6 ligand) was purchased from Molecular Probes. Gardiquomod (TLR 7/8 ligand) was purchased from InvivoGen ELISA kits for human cytokines and NF-{kappa}B (p65) were purchased from BioSource International.

Cell preparation and culture

Heparinized blood was drawn from healthy normal volunteers after informed consent was obtained. Human PBMC were isolated by Ficoll-Paque (Amersham Biosciences) density gradient centrifugation and resuspended in RPMI 1640 medium with 10% FBS. Adherent monocytes were obtained by incubating PBMC in RPMI 1640 medium for 2 h at 37°C in a humidified 5% CO2 and 95% air atmosphere. The nonadherent cells were removed, and adherent monocytes were cultured for further study. Immature DC were generated from CD14+ monocytes, as in our previous studies (19).

TLR ligand stimulation

PBMC or immature DC obtained above were incubated for 48 h in RPMI 1640 containing 1% FCS, at 5 x 105/ml in 12 well plates (1 ml/well), with or without varying concentrations of gangliosides. During the last 24 h of incubation, TLR ligands (LTA, LPS, poly(I:C), zymosan A, and gardiquimod) were added to the cultures. At the end of the incubation, culture supernatants were harvested for cytokine (IL-6, IL-12, and TNF-a) quantification.

For nuclear protein extraction, immature DC were incubated with 50 µM GD1a for 48 h and/or TLR ligands (LTA, LPS, poly(I:C), and zymosan A) for the last 24 h, and monocytes were treated with 50 µM GM1 for 24 h followed by stimulation with LPS for up to 24 h. After incubation, cells were harvested and washed twice in cold PBS. The cell pellet was resuspended in 100 µl of lysis buffer (10 mM HEPES (pH 7.9), 1 mM EDTA (pH 8.0), 150 mM NaCl, 1 mM DTT, 1 mM PMSF, 0.5 mM sodium vanadate, and 1% Nonidet P-40) in 1.5 ml Eppendorf tubes for 20 min on ice and then centrifuged at 12,000 rpm at 4°C for 15 min to pellet the nuclei, and the supernatant containing the cytoplasmic protein removed. The pellet was resuspended in 50 µl of nuclear lysis buffer (10 mM HEPES (pH 7.9), 5 mM EDTA (pH 8.0), 150 mM KCl, 0.05% SDS, 1% triton, 20 mM NaF, 20 mM sodium pyrophosphate, 20 mM B-glycerophosphate, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM DTT), incubated on ice for 15 min and freeze-thawed three times. These nuclear extracts were collected for NF-{kappa}B (p65) quantification by ELISA.

For the preincubation studies, monocytes were incubated with 0.1 µg/ml LPS for 24 h (control) or preincubated 50 µM GM1 for varying periods (0–24 h), following which 0.1 µg/ml LPS was added to the ganglioside-containing cultures for an additional 24 h, before cytokine quantification. For the washout studies, PBMC were incubated with 50 µM GD1a for 24 h, then medium containing unbound gangliosides was removed, and cells were washed with medium twice. A total of 0.1 µg/ml LPS was added into the culture after 0–24 h and cells were cultured for another 24 h in medium with 1% FCS. At the end of the culture period, all supernatants were harvested for cytokine (IL-6, IL-12, and TNF-{alpha}) quantification by ELISA.

ELISA

Using the BioSource kit protocol, ELISAs were performed for cytokines IL-6, TNF-{alpha}, and IL-12 on the supernatants harvested from PBMC and monocytes, and for NF-{kappa}B (p65) measurement on nuclear extracts from monocytes and DC. Data obtained from the ELISA were expressed as absolute quantities, comparing the effect of ganglioside treatment vs control cultures.

Detection of IRAK-M expression

Monocytes were incubated with 0.1 µg/ml LPS or 50 µM purified GM1 for 24 h to assess IRAK-M expression. Cells were harvested and washed twice in cold PBS. Cell cytoplasmic protein was extracted as described above and quantified. A total of 20 µl of cell lysate from each sample was mixed with (4x) NuPAGE LDS Sample buffer (Invitrogen Life Technologies) and boiled for 5 min. After electrophoresis on 4–12% NuPage Bis-Tris precast gel (Invitrogen Life Technologies), the proteins were transferred to Immobilon P membranes (Millipore). The membranes were blocked in freshly prepared PBS, containing 3% dry milk, then were incubated with 0.5 µg/ml of polyclonal anti-IRAK-M Ab (Chemicon International) overnight at 4°C, followed by washing three times with PBS containing 0.05% Tween 20. The membranes were incubated with a horseradish-peroxidase-conjugated goat anti-rabbit Ig (Ig)-G (1/8000) (Sigma-Aldrich) in PBS containing 3% dry milk for 1 h at room temperature with agitation. After washing three times with PBS-0.05% Tween 20, the membranes were incubated with Super Signal Chemiluminescent Substrate Stable Peroxide solution (Pierce) for 5 min and exposed to Biomax-MR film (Eastman Kodak).

Statistical analysis

Results are reported as the mean ± SEM of 2–3 separate experiments, as indicated. The significance of differences between the experimental data and control data was determined using Student’s paired-comparison t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Since the exogenous addition of ganglioside GD1a inhibits activation of monocytes by LPS (20), we assayed the effect of exogenous gangliosides on activation of other TLRs, as assessed by the activation of proinflammatory cytokine production. To begin to determine optimal concentrations, we developed a concentration-response curve for GD1a. Human PBMCs were preincubated with a range of concentrations of ganglioside GD1a, for 24 h, then stimulated with LPS (TLR4 ligand), LTA (TLR2 ligand), or medium alone as a negative control. Preincubation of PBMCs with GD1a potently inhibits LPS- and LTA-induced IL-12 (Fig. 1A) and TNF-{alpha} (Fig. 1B) expression as measured by release into the culture supernatants, with maximal inhibition observed at a ganglioside concentration of 20–50 µM. To determine whether inhibitory effects of GD1a exposure might be caused by increased apoptosis of the cells, we exposed unstimulated PBMC to 50 µM GD1a in medium with 2% FCS for 24 or 48 h and assessed apoptosis by flow cytometry using 7-ADD and Annexin V staining. Exposure of PBMC to GD1a did not cause increased apoptosis of the cells (12.7 ± 0.1% vs 10.6 ± 0.2% at 24 h and 17.2 ± 0.3% vs 16.1 ± 2.0% at 48 h, control vs GD1a exposed, respectively). These findings, together with the complete reversibility of inhibition of DC function by removal of the ganglioside (vide infra), are consistent with past findings (17, 19) excluding a nonspecific toxic effect of ganglioside exposure.


Figure 1
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  		FIGURE 1. Effect of GD1a ganglioside exposure on cytokine production by TLR receptor-stimulated PBMCs. Human PBMCs were cultured for 24 h with media or ganglioside GD1a at 5, 20, or 50 µM, after which, medium, 0.1 µg/ml LPS, or 50 µg/ml LTA was added during an additional 24-h incubation. The culture supernatants from control and stimulated PBMCs were harvested and assayed by ELISA for IL-12 (A) and TNF-{alpha} (B). Key: *, p < 0.05 and **, p < 0.01, control vs ganglioside incubations. Results are representative of three separate experiments.

 
Next, we tested the effect of exogenous gangliosides on the activation of monocyte or PBMC TLRs by a range of ligands. These included poly(I:C) (TLR3 ligand), zymosan A (TLR2/TLR6 ligand), LTA (TLR2 ligand), and LPS (TLR4 ligand). Preincubation of PBMCs with 50 µM GD1a potently inhibited activation by all of these TLR ligands, indicating that ganglioside treatment affects TLRs located on the plasma membrane of the cell (Fig. 2, A–C). Preincubation with GD1a also inhibited IL-12 expression following stimulation with gardiquimod, a TLR 7/8 ligand (Fig. 2D), although expression level of IL-12 was low in the control, consistent with a low level of expression of TLR7/8 on PBMCs (40, 41). Since TLR7 and TLR8, like TLR3, are stimulated in the endosomal compartment, this latter finding suggests that TLR signaling within endosomes is also inhibited by exogenous gangliosides. Preincubation with GD1a had no effect on PMA/ionomycin-induced cytokine release by PBMC (data not shown), indicating that the effect is specific to TLR signaling.


Figure 2
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  		FIGURE 2. Effect of GD1a exposure on plasma membrane and endosomal TLR ligand-induced cytokine production by PBMCs. Human PBMC were cultured with or without 50 µM GD1a for 48 h and with TLR ligands during the last 24 h of the incubation. Then, the culture supernatants were harvested for cytokine (IL-6, IL-12, and TNF-{alpha}) quantification with ELISA. A–C, Cytokine production in response to the plasma membrane TLR ligands LTA (50 µg/ml), LPS (0.1 µg/ml), poly(I:C) (50 µg/ml), and zymosan A (12.5 µg/ml). D, IL-12 production in response to the endosomal TLR ligand Gardiquomod (1 µg/ml). **, p < 0.01 in comparison to nonganglioside incubation.

 
We then tested several gangliosides to determine optimal inhibitory concentrations and possible differential effects that might depend on structural differences among different specific gangliosides. Human PBMCs were preincubated with increasing concentrations of gangliosides GD1b, GM1, and GM3 for 24 h, then stimulated with the LPS (TLR4 ligand), LTA (TLR2 ligand), or medium alone as a negative control. Preincubation of PBMCs with GD1b and GM1 potently inhibited LPS- and LTA-induced IL-12 (Fig. 3, A and B) and TNF-{alpha} (Fig. 3, C and D) (similar to the effect of GD1a) whereas GM3, the simplest of the gangliosides tested, had no inhibitory effect (Fig. 3, E and F). GM1 inhibited activation somewhat more than did GD1a or GD1b, and maximal inhibition by all three gangliosides was at a 20–50 µM concentration.


Figure 3
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  		FIGURE 3. Effect of ganglioside concentration on cytokine release from TLR ligand-stimulated PBMCs. Human PBMCs were cultured with purified gangliosides (0–50 µM GD1b or GM1, or 0–100 µM GM3) for 24 h, after which, TLR ligands LPS or LTA were added to the cultures for an additional 24 h. After incubation, supernatants of the cultures were collected for cytokine IL-12 (A, C, and E) and TNF-{alpha} (B, D, and F) quantification by ELISA. A and B, 0–50 µM GD1b; C and D, 0–50 µM GM1; and E and F, 0–100 µM GM3. Results are representative of three separate experiments.

 
Stimulation of proinflammatory cytokine expression via the TLR family is mediated in part by nuclear translocation of the proinflammatory transcription factor, NF-{kappa}B. Previously, we observed that pre-exposure of GD1a inhibited the nuclear binding activity of NF-{kappa}B of LPS-induced immature DC (19). In this study, we also show that activation of DC by four of the TLR ligands used in the present experiments (LTA, poly(I:C), LPS, and zymosan A) induced expression of NF-{kappa}B protein (p65) in the nuclear fraction within 1 h, and that it persisted for up to 24 h. In contrast, membrane enrichment of DC with ganglioside GD1a caused a substantial reduction in the NF-{kappa}B nuclear expression that is normally observed as a response to TLR ligand stimulation (Fig. 4A). We also compared the time course of NF-{kappa}B translocation following exposure of monocytes either to the TLR ligand LPS or to the ganglioside GM1 (Fig. 4B). NF-{kappa}B translocation occurs at a very early stage of ligand stimulation, being at near maximal levels at 1 h, and then lasting for 24 h. The absence of nuclear translocation of NF-{kappa}B in ganglioside-enriched cells was observed over the entire time course of ganglioside exposure, suggesting that the observed absence of translocation in our studies is not merely explained by a change in time kinetics and therefore that ganglioside exposure does not induce NF-{kappa}B nuclear translocation.


Figure 4
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  		FIGURE 4. Effect of GD1a on nuclear expression of NF-{kappa}B protein (p65) in TLR ligand-stimulated human DC and monocytes. A, Human monocyte-derived dendritic cells were cultured with or without 50 µM GD1a for 48 h, with various TLR ligands (LTA, LPS, poly(I:C), and zymosan A) added individually to the cultures for the last 24 h. B, Time course study: Monocytes were cultured with or without 50 µM GM1 for 24 h, then stimulated with 0.1 µg/ml LPS for 0–24 h with GM1 present in the culture. After incubation, nuclear proteins were extracted, protein concentration was equalized, and NF-{kappa}B (p65) was measured by ELISA. Key: *, p < 0.05 and **, p < 0.01, control vs ganglioside incubations.

 
Having established that a subset of gangliosides inhibits TLR ligand-induced up-regulation of proinflammatory cytokine production, we next determined how extensive a preincubation with ganglioside was necessary to result in this inhibition. As shown in Fig. 5A, even without preincubation, the presence of added ganglioside at the time of stimulation by the TLR ligand LPS resulted in almost complete abrogation of cytokine release during the subsequent 24-h culture period, suggesting that a rapid interaction is responsible. We also assessed whether or not inhibition requires the presence of the exogenous gangliosides in the culture medium. PBMCs were incubated for 24 h with 50 µM GD1a. Then, the cells were washed to remove exogenous ganglioside and incubated for varying periods of time before stimulation with LPS. When LPS was added immediately after removal of exogenous ganglioside (0 h), complete inhibition of TLR signaling was observed. However, within an hour of removal of ganglioside from media, LPS-stimulated PBMC cytokine release began and there was complete recovery by 6–24 h. The results suggest that inhibition of TLR signaling caused by exogenous gangliosides is rapid and is reversible after gangliosides are removed from the culture (Fig. 5, B and C). Also, the latter finding further eliminates a cytotoxic effect of the ganglioside as a cause for the inhibition we observed (17, 19).


Figure 5
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  		FIGURE 5. Inhibition of LPS-stimulated DC cytokine release: effect of time of ganglioside preincubation and reversibility by ganglioside washout. A, Monocytes were incubated with 0.1 µg/ml LPS for 24 h (control) or preincubated 50 µM GM1 for varying periods (0–24 h), following which 0.1 µg/ml LPS was added to the ganglioside-containing cultures for an additional 24 h and then the supernatants of the cultures were collected for cytokine (IL-12, IL-6, and TNF-{alpha}) quantification by ELISA. The control was incubation with LPS alone without ganglioside addition for 24 h. Key: **, p < 0.01, LPS-stimulated vs ganglioside exposed, LPS-stimulated cultures. Results are representative of three separate experiments. B and C, PBMC were cultured with 50 µM GD1a or medium for 24 h, washed twice to remove unbound GD1a, and cultured in control medium for 0–6 h after the ganglioside washout. Then, LPS in fresh medium (without GD1a) was added to the cultures for another 24 h and the supernatants collected for quantification of TNF-{alpha} (B) and IL-6 (C). Key: M = medium only; LPS = 0.1 µg/ml for 24 h (no GD1a exposure); LPS plus GD1a = 50 µM GD1a for 48 h, and 0.1 µg/ml LPS for the last 24 h; washout = GD1a (50 µM) for 24 h, followed by washout, 0–6-h incubation in medium, and then by 0.1 µg/ml LPS for 24 h. Results are representative of two separate experiments. Key: *, p < 0.05 and **, p < 0.01, LPS-stimulated vs ganglioside exposed, LPS-stimulated cultures.

 
The transient inhibition of TLR signaling is similar to the in vitro endotoxin tolerance phenotype, whereby treatment of monocytes with LPS leads to a refractory period during which TLRs do not respond to TLR ligands, analogous to sepsis-induced immunosuppression. One mediator of the endotoxin tolerance phenotype is the TLR pathway inhibitor IRAK-M (37); IRAK-M is up-regulated within 24 h of LPS treatment, endotoxin tolerance is abolished in the absence of its expression in IRAK-M knockout mice (37), and peptidoglycan tolerance can be abolished in human monocytes using IRAK-M short interfering RNA (siRNA) (42). IRAK-M has also been reported to be involved in tumor-induced tolerance (43). As a result of these similarities between endotoxin and peptidoglycan tolerance and ganglioside enrichment in inhibiting cytokine production, we investigated IRAK-M expression by Western blotting following ganglioside treatment. Peripheral blood monocytes were treated with purified GM1 for 24 h, with medium alone serving as a negative control and LPS treatment as a positive control. The cells were then harvested and lysed for Western blotting for IRAK-M expression, and the supernatants harvested for cytokine measurement. Treatment with ganglioside GM1 alone induced IRAK-M expression at a level similar to that seen following treatment with LPS for 24 h (Fig. 6, A and B). However, in contrast to the initial proinflammatory effect of LPS treatment, with an increased expression of proinflammatory cytokines, ganglioside GM1 treatment alone did not stimulate the expression of any of the three proinflammatory cytokines assayed (Fig. 6C).


Figure 6
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  		FIGURE 6. IRAK-M expression and proinflammatory cytokine release by monocytes treated with LPS or ganglioside. Monocytes were incubated with media, 0.1 µg/ml LPS, or 50 µM of GM1 for 24 h and then harvested and lysed to extract cytoplasmic protein for Western blotting, and the culture supernatant was analyzed by ELISA for cytokine levels. A, Immunoblotting for IRAK-M and GAPDH protein (in 20 µg of total cell lysate). B, Relative fold increase in levels of expression of IRAK-M, normalized to GAPDH by densitometry. C, Cytokine levels (TNF-{alpha}, IL-6, and IL-12) in culture supernatants. Results are representative of three independent experiments. Key: **, p < 0.01, LPS-stimulated vs ganglioside-incubated cultures.

 
We also evaluated the timing of IRAK-M expression induced by monocyte exposure to LPS or GM1 (Fig. 7). IRAK-M expression was detected somewhat earlier in the LPS-stimulated monocytes (clearly seen at 3 h) than in those exposed only to GM1 ganglioside (clearly seen at 6 h). Again, robust detection was observed at 24 h in both sets of cultures.


Figure 7
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  		FIGURE 7. Time course of LPS- or ganglioside-induced monocyte IRAK-M expression. Monocytes were incubated with 0.1 µg/ml LPS (A) or 50 µM GM1 (B) for varying times (1, 3, 6, and 24 h), harvested, lysed, and IRAK-M expression detected by immunoblotting with anti-IRAK-M Ab. Results are representative of two independent experiments.

 
In conclusion, exogenous ganglioside exposure appears to induce the expression of the inhibitory IRAK-M but, unlike TLR ligands, does not induce the production and release of proinflammatory cytokines by the APC.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Immune surveillance provides the host with the ability to limit the growth and progression of tumors. However, tumors appear to possess several mechanisms to limit immune responses (44). These include loss of HLA expression, absence of tumor Ag expression, defective or deficient apoptotic pathways, and production of immunosuppressive factors (45). These latter tumor-derived molecules have the potential to create local and systemic immunosuppression, leading to evasion of immune responses with progression of disease, and may limit the efficacy of immune-based therapies. Identification and thorough characterization of the mechanisms of immune evasion is critical for the development of effective immune-based therapies for cancer, as recent successes continue to provide hope that immune-based therapies will eventually be a feasible treatment strategy for many cancers (46).

Gangliosides are immunosuppressive tumor-derived molecules (1, 13). They are present in the outer leaflet of all plasma membranes, with the highest concentrations being present in the CNS. They also are abundant in certain types of tumors, particularly tumors of neuroectodermal origin, which exhibit tumor-specific ganglioside expression profiles (5). Tumor cells shed these molecules at a high rate into their local microenvironment, and highly efficient transfer of shed gangliosides from tumor cells to target cells has been documented (47), an important point with respect to the biological activity of tumor gangliosides. Although accurate measurements of ganglioside concentrations in the tumor microenvironment have not yet been possible, significant shedding into the peripheral circulation in humans is well known, resulting in elevated levels in the serum of some patients with cancer (5). Interestingly, abnormalities in ganglioside expression have also been demonstrated in certain immunologic diseases, specifically in T cells from patients with systemic lupus erythematosus (15). Activated T cells from these patients have enhanced lipid raft formation with enhanced expression of ganglioside GM1, which may play an important role in the pathogenesis of systemic lupus erythematosus (48, 49).

The current study demonstrates that exogenous gangliosides potently suppress effective TLR activation in a dose-dependent manner. The inhibitory effect of ganglioside enrichment of monocytes is reversible, with complete recovery of effective TLR signaling, as assessed by proinflammatory cytokine production, within 24 h of removal of gangliosides from the culture medium and without an effect on cell count or cell viability, or an increased degree of apoptosis. These observations suggest that there is no irreversible toxicity of the gangliosides during the 48-h exposure we used, in contrast to a reported apoptotic effect of a longer (6 day) GM3 or GD3 ganglioside exposure of monocytes that were being treated with GM-CSF and IL-4 to cause differentiation into DC (50). Prolonged exposure may be required to reveal proapoptotic properties of these molecules, whereas the effects observed in the current study appear to be direct effects on signaling through TLRs.

A further clue toward identification of the mechanism by which membrane ganglioside enrichment acts lies in the observation that gangliosides in the media did not appear to inhibit the binding of TLR ligands to their receptors. This is supported by our findings that exogenous gangliosides could be removed from the culture medium before LPS treatment without substantially reversing the inhibition of proinflammatory cytokine production caused by the ganglioside exposure. It is possible that ganglioside enrichment could have an effect on TLR receptor conformation, leading to a decreased receptor affinity for ligand. In the case of flagellin binding to TLR5, however, exogenous addition of gangliosides inhibited TLR5 signaling induced by flagellin but did not reduce binding of flagellin to the receptor (23).

A differential effect of specific gangliosides on APC differentiation has been previously reported. Incubation of human monocytes with GM2 inhibits the differentiation of DC in the presence of cytokines, as demonstrated by enhanced adherence and cell spreading with reduced expression of MHC class II and costimulatory molecules; in contrast, GM3 had no effect (51). In a more recent study looking at the effects of human milk-derived gangliosides, GD3 inhibited maturation of bone marrow-derived DC in response to LPS, as measured by diminished production of proinflammatory cytokines and reduced proliferation in a MLR, whereas GM3 had lesser effects (52). In polarized T cells, GM3 and GM1 are localized at opposite poles (53). Localization and organization of gangliosides in membrane microdomains of APCs is not characterized but could explain the differential effects. Alternatively, the varied spatial structures of the gangliosides in the membrane may differentially affect the conformation of cell surface protein.

The molecular mechanisms of immunosuppression by exogenous gangliosides remain to be fully elucidated. Many receptors demonstrate altered signaling following ganglioside treatment (54). For example, epidermal growth factor receptors are sensitized to activation by ganglioside-induced ligand independent receptor dimerization (55), whereas, as we found here, TLR signaling is inhibited. As a result of the variable effects on many distinct signaling systems, it seems most probable that the mechanism involves a generalized alteration of the membrane when these amphipathic molecules insert into membranes, rather than activation (or inhibition) of a specific receptor. Interestingly, TLR activation leads to translocation of the TLR from a mobile fraction to an immobile fraction with characteristics of lipid islands (29). It is possible that exogenous gangliosides disrupt or alter this translocation and thus inhibit activation of the pathway, although supportive data for this model are still lacking.

It is interesting that the phenotype of ganglioside-treated monocytes is identical with that of the in vivo endotoxin tolerance phenotype, or sepsis-associated immunosuppression (56). The in vitro correlate of endotoxin tolerance is the failure to stimulate TLR activation during a window of up to 24 h after initial stimulation with TLR ligand. One mechanism for endotoxin tolerance appears to be the rapid up-regulation of the TLR pathway inhibitor, IRAK-M, upon restimulation of monocytes with LPS following a previous LPS treatment (37, 57). IRAK-M expression is restricted to the monocytic lineage. Using IRAK-M knockout mice and siRNA to suppress IRAK-M up-regulation, the endotoxin tolerance phenotype to LPS and peptidoglycan have been shown to be dependent upon IRAK-M expression (37, 42). We therefore measured IRAK-M protein expression after ganglioside treatment of naive monocytes and found that the exogenous ganglioside GM1 (as well as GD1a, unpublished observations) up-regulates IRAK-M expression. Importantly, however, preincubation of monocytes with gangliosides does not induce cytokine release or NF-{kappa}B nuclear translocation but does up-regulate expression of the TLR pathway inhibitor, IRAK-M, with a time course and level of expression similar to that seen with LPS treatment.

In this study, we found that ganglioside GM1 alone induced IRAK-M expression, identical with effects of TLR ligands, but without initiating downstream NF-{kappa}B activation and proinflammatory cytokine release. Our data and those of others, e.g., that TLR5-flagellin binding is intact in the presence of added gangliosides (23), suggest that exogenous gangliosides neither inhibit binding of ligands to TLRs nor inhibit the initial activation steps but bypass downstream activation steps that occur before NF{kappa}B activation and lead directly to IRAK-M expression. The nature of this novel inhibitory mechanism remains to be identified, while its elucidation may also be relevant to addressing the question of whether ganglioside expression could play an important role in the mechanism of endotoxin tolerance, as has been suggested (58).

In cancer, other tumor-derived molecules may have a somewhat similar activity to that which we have shown here for gangliosides. For example, hyaluronan, which is a normal component of the extracellular matrix, appears to stabilize TLRs in an inactive state when it is present in the polymerized form, whereas when it is degraded into smaller fragments (such as in the setting of lung damage), it activates TLRs (59). A recent study demonstrates that coincubation of tumor cell lines with monocytes inhibits activation of the monocytes, proposed to be due to tumor cell-derived hyaluronan, acting through TLR4 and CD44 (43). Interestingly, treatment of monocytes with purified hyaluronan initially activates TLRs, causing expression of proinflammatory cytokines, but subsequently leads to inhibition of activation associated with up-regulation of IRAK-M. Suppression of the up-regulation of IRAK-M by siRNA inhibits the inhibitory phenotype, demonstrating the necessity of IRAK-M for the inhibitory phenotype. In contrast to the effects of hyaluronan (which like LPS leads to proinflammatory cytokine expression and then IRAK-M up-regulation), gangliosides do not stimulate cytokine production.

In conclusion, exogenous gangliosides potently inhibit TLR signaling through multiple TLRs in a manner dependent both on the concentration of the ganglioside and its structure. The inhibitory phenotype is reversible, with recovery of TLR signaling after removal of exogenous gangliosides occurring within 24 h. Up-regulation of IRAK-M by gangliosides is likely to be involved in the tumor ganglioside-induced suppressive effects on APC function, underscoring an important role these tumor-derived molecules may play in subverting host antitumor immune responses.


   Acknowledgments
 
We thank Arlene Gendron and Melva McGlen for preparation of the manuscript.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 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 Grant R01 CA42361 (to S.L.) and by The Children’s Cancer Foundation. K.D.S. is the recipient of an National Institute of Child Health and Human Development Child Health Research Scholar Award (K12 HD001399). Back

2 W.S. and K.S. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Stephan Ladisch, Center for Cancer and Immunology Research, Children’s National Medical Center, 111 Michigan Avenue NW, Washington, DC 20010. E-mail address: sladisch{at}cnmc.org Back

4 Abbreviations used in this paper: DC, dendritic cell; IRAK-M, interleukin-1 receptor associated kinase-M; LTA, lipoteichoic acid; poly(I:C), poly (cytidylic-inosinic) acid; siRNA, short interfering RNA. Back

Received for publication April 7, 2007. Accepted for publication January 25, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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