Infectious agents, including bacteria and viruses, are thought to provide triggers for the development or exacerbation of autoimmune diseases such as systemic lupus erythematosus in the genetically predisposed individual. Molecular mimicry and engagement of TLRs have been assigned limited roles that link infection to autoimmunity, but additional mechanisms are suspected to be involved. In this study we show that T cells from lupus-prone mice display aggregated lipid rafts that harbor signaling, costimulatory, inflammatory, adhesion, and TLR molecules. The percentage of T cells with clustered lipid rafts increases with age and peaks before the development of lupus pathology. We show that cholera toxin B, a component of Vibrio cholerae, promotes autoantibody production and glomerulonephritis in lupus-prone mice by enhancing lipid raft aggregation in T cells. In contrast, disruption of lipid raft aggregation results in delay of disease pathology. Our results demonstrate that lipid rafts contribute significantly to the pathogenesis of lupus and provide a novel mechanism whereby aggregated lipid rafts represent a potential link between infection and autoimmunity.
Genetic and environmental factors contribute to the initiation and development of autoimmune diseases (1). Infectious agents, including bacteria and viruses, provide triggers for the initiation or exacerbation of autoimmune diseases in the genetically predisposed individual (2). Molecular mimicry and engagement of TLR have been assigned mechanistic roles whereby infectious agents instigate autoimmune diseases (3, 4). Molecular mimicry refers to shared structural homology between infectious agent components and proteins of the host (3). However, despite the fact that several infectious agents have been reported to promote autoimmune diseases, the number of identified molecular mimicry cases is quite limited (2). TLRs are key components of the innate immune system and crucial regulators of both innate and adaptive immune responses. Autoimmune disease may be enhanced by several TLR ligands in mouse models, but TLR-dependent processes do not always explain the development of autoimmunity (4). The determination of additional mechanisms whereby pathogens provoke or promote autoimmune disease should be of scientific and clinical value.
In recent years, numerous studies demonstrated that a wide range of infectious agents, including bacteria, viruses, parasites, and prions, infect mammalian cells only through intact lipid rafts (5, 6, 7, 8). Lipid rafts have been shown to be involved in various cell processes, including pathogen internalization, intracellular maturation of phagosomes, lysis and fusion of phagosomes, activation of intracellular signaling molecules, induction of cell death following infection, and release of cytokines (7, 8).
Lipid rafts are enriched in sphingolipid and cholesterol microdomains on plasma membranes and serve as platforms that bring together signal proteins. As such, they are pivotal in immune cell receptor-initiated signaling and its regulation both in terms of strength and duration (9). Besides the immune receptors that engage Ag on T, B, and NK cells, a number of costimulatory molecules become invariable components of the lipid rafts and these include MHC class II, CD40, CD95, CD28, CTLA-4, and FcγRIIB1 (9, 10, 11). Lipid rafts are crucial in the regulation of TCR signaling (9).
Systemic lupus erythematosus (SLE)3 is a multisystem autoimmune disease characterized by inflammatory damage of various organs including the kidney and the skin, the production of autoantibodies against nuclear Ags, and abnormalities in T cell function and receptor signaling (12). Current evidence suggests that T cells have an important role in the pathogenesis of SLE (13). Clustered lipid rafts have been found on the surface membrane of T cells from patients with SLE and shown to contribute to the aberrant CD3-mediated signaling (14, 15). Yet, it is unclear whether lipid rafts contribute to the pathogenesis of SLE. Patients with SLE are more prone to suffer various infections that, in turn, may enhance disease activity, and infections remain a major cause of morbidity and mortality in SLE (16). However, the mechanisms whereby infections exacerbate SLE pathology remain unclear.
In this study we show that Vibrio cholerae cholera toxin B (CTB) promotes disease progression in lupus-prone mice by enhancing T cell lipid raft aggregation, whereas disruption of lipid rafts delays disease progression. Clustered lipid rafts on T cells in MRL/lpr mice were found to contain diverse molecules, including TCR signaling, inflammatory, costimulatory, adhesion, and TLR molecules. The costimulatory T cell response mediated by these molecules following TCR ligation depends on the presence of intact lipid rafts. Our data strongly suggest that lipid rafts provide a potential link between infectious agents and autoimmune disease.
Materials and Methods
Mice and materials
Female MRL/lpr/2J mice, (New Zealand Black × New Zealand White)F1 (NZB/W F1) mice, MRL/MpJ mice, B6.MRL/lpr mice, CD40 ligand (CD40L) knockout mice, and C57BL/6 (B6) mice were purchased from The Jackson Laboratory and housed in the animal facility of Beth Israel Deaconess Medical Center. Methyl-β-cyclodextrin (MβCD), α-cyclodextrin (αCD), CTB, FITC-CTB, anti-actin Ab, dsDNA, mouse IgG Ab, mouse complement 3 (C3) Ab, and cytochalasin D were purchased from Sigma-Aldrich. Abs to TNFR2, IFN-γ, MCP-1, TNF-α, CD4, and CD8 for immunofluorescent staining were purchased from Santa Cruz Biotechnology. Ab of CD40L and TNFR2, recombinant TNF-α, and MCP-1 were purchased from R & D Systems. Syk and CD44 Abs were purchased from Abcam. Abs to TLR4 and TLR9 were purchased from Santa Cruz Biotechnology.
T cells were isolated from spleens of MRL/lpr mice or B6 mice or other mouse strains using mouse T cell enrichment columns (R&D Systems). T cells were fixed with 3% paraformaldehyde in PBS and cytospun onto slides, permeabilized with 0.05% Triton X-100 for 5 min at room temperature, and blocked with PBS containing 10% normal goat serum. Primary Abs in 0.5% BSA was incubated for 45 min at room temperature. Cells were washed and incubated with a secondary Ab and FITC-conjugated CTB for 45 min. After three PBS washes, nuclei were stained with 4′,6′-diamidino-2-phenylindole for 5 min. Slides were mounted with a coverslip using Fluoromount-G (SouthernBiotech). T cells were examined using a Zeiss LSM 510 META confocal microscope. For enumeration of the percentage of cells with FITC-conjugated CTB, 250–500 cells were scored visually by a single blinded observer. Cells in which staining with CTB-FITC did not display a homogenous ring staining pattern and displayed at least one aggregate covering one-eighth of the cell circumference were identified as cells with aggregated lipid rafts.
Treatment of MRL/lpr mice by MβCD and CTB
Female MRL/lpr mice received MβCD (1 mg/mouse, i.p., n = 12) or PBS (n = 12) three times a week starting at age of 4 wk for 16 wk. Five MRL/lpr mice in each group were sacrificed at the age of 16 wk. The remaining mice were followed for survival to the end of the experimental period. Kidney and serum from sacrificed mice were collected for histological examination and the measurement of serum IgG and anti-dsDNA Ab. Female MRL/lpr mice were treated i.p. with either CTB (2 μg/mouse, n = 8) or PBS (n = 8) once a week starting at the age of 4 wk for 12 wk or MβCD (1 mg/mouse) three times per week starting at age of 4 wk for 4 mo. Mice were sacrificed at end of the experiment and samples of serum and kidney were collected for examination. During the experimental period, urine protein content and mortality were monitored.
Kidney examination of histology and immunofluorescence
Kidneys were fixed with 10% buffered formalin, embedded in paraffin, and sectioned into 4-μm pieces. Kidney tissues were stained with either the periodic acid-Schiff (PAS) reagent or with H&E. The slides were coded and blindly assessed. Renal pathology is graded by glomerular, interstitial, and perivascular inflammation. Scores from 0 to 4 are assigned for each of the features. A minimum of 100 glomeruli were assessed to determine the glomerular scoring index in each mouse. For kidney immunofluorescence examination, cryostat-sectioned tissues were stained with fluorescein-conjugated Abs detecting mouse IgG or mouse C3. Sections were examined with confocal microscopy.
The mice in each group were placed overnight in a Nalgene metabolic cage to collect urine. Urine was measured with Multistix 10 SG reagent strips and analyzed by Clinitek Status analyzer (Bayer Healthcare). Proteinuria is expressed as follows: 0, none; 1, 30–100 mg/dl; 2, 100–300 mg/dl; 3, 300-2000 mg/dl; or 4, >2000 mg/dl.
Measurement of serum IgG and anti-DNA Ab
Serum IgG and anti-dsDNA Abs were detected by ELISA. A 96-well plate was coated with double calf thymus DNA (1.5 mg/ml; Sigma-Aldrich) or mouse IgG Ab (0.1 mg/ml; Sigma-Aldrich) in PBS at 4°C overnight. After blocking with 1% BSA for 2 h at room temperature, serum samples were added to the plate and incubated for 2 h at room temperature. After washing four times with PBS, HRP-conjugated goat anti-mouse IgG Ab (Sigma-Aldrich) was added to the plate and incubated for 1 h at room temperature. Ab binding was visualized using 3′,3′,5′,5′-tetramethylbenzidine (Sigma-Aldrich), and plates were read with the OD at 450 nm.
T cell proliferation assay
T cells (1 × 105) isolated from the spleens of B6 mice were stimulated with anti-CD3 Ab (0.1 μg/ml) in the presence or absence of CD28 Ab (10 μg/ml), TNF-α (20 ng/ml) or TNFR2 Ab (10 μg/ml), or MCP-1 (2 μg/ml) with or without MβCD (20 mg/ml) and cultured for 72 h at 37°C. After 60 h, cultures were pulsed with [3H]thymidine (0.5 μCi/10 μl/well) for 12 additional hours. Data presented are mean cpm ± SEM of responses from three replicate cultures. IFN-γ levels in culture supernatants were measured using a mouse IFN-γ ELISA kit (R&D Systems).
Statistical evaluations of clustered lipid rafts, renal pathology, serum IgG, anti-dsDNA Ab, T cell proliferation and IFN-γ data were performed using the Student’s t test. p ≤ 0.05 was considered statistically significant. Survival was assessed using Kaplan-Meier survival and long rank test.
CTB promotes disease progression in MRL/lpr mice
After we found that lipid rafts are aggregated on the surface membrane of T cells from patients with SLE and demonstrated that they contribute to increased CD3-mediated signaling responses (14) we wished to determine whether lipid raft clustering contributes to the pathogenesis of disease. CTB, a nontoxic component of V. cholerae, binds directly to cell surface gangliosides (GM1) (17, 18) present in lipid rafts and mediates lipid raft aggregation (18, 19). Accordingly, we considered that if lipid raft clustering contributes to disease progression, treatment of lupus-prone mice with CTB should accelerate the appearance of autoimmunity and related pathology. MRL/lpr mice, a well established animal model of SLE, were treated weekly with CTB (2 μg/mouse i.p.) starting at the age of 4 wk for 12 wk. Whereas, three mice died in the CTB-treated group, all mice in the control PBS-treated group remained alive (Fig. 1,A; p = 0.08). There was increased proteinuria, hematuria, numbers of leukocytes in urine, and levels of serum IgG and anti-DNA Ab titers in mice treated with CTB compared with the control group (Fig. 1, B–F). Kidney pathology was significantly more severe in CTB-treated mice compared with control mice (Fig. 1, G–H). We noted increased IgG and C3 deposition in glomeruli of mice treated with CTB compared with glomeruli of mice treated with PBS (Fig. 1,I). We also observed that lung and liver injury was significantly more severe in CTB-treated mice compared with control mice (Fig. 1 J). We did not observe a clear effect of CTB on the sizes of lymph nodes and spleen in the treated mice. These data demonstrate that CTB promotes disease severity in MRL/lpr mice.
Lipid raft clustering on T cells from MRL/lpr mice
To understand how CTB promotes disease progression in MRL/lpr mice, we first determined whether T cells from MRL/lpr mice display aggregated lipid rafts in a manner similar to that seen in human SLE T cells (14, 15). T cells isolated from spleens of MRL/lpr mice and B6 mice were stained with FITC-CTB and the lipid raft distribution on the surface membrane was studied using confocal microscopy. Increased numbers of T cells from MRL/lpr mice displayed aggregated lipid rafts on the surface membrane compared with similarly stained T cells from normal B6 mice (Fig. 2 A).
To determine the kinetics of appearance of lipid raft clustering, T cells from MRL/lpr mice were examined at different time points. We found that T cells with aggregated lipid rafts appeared in 3-wk-old mice and peaked by the age of 6–7 wk (Fig. 2,B). To determine whether the appearance of T cells with clustered lipid rafts correlates with lupus pathology in MRL/lpr mice, we performed renal histology at different time points. We found that the appearance of T cells with clustered lipid rafts preceded the development of renal pathology. Eight-week-old mice did not display glomerulonephritis, whereas glomerulonephritis was prevalent in 16-wk-old mice (not shown). There was a minimal (<5%) percentage of T cells with clustered lipid rafts on T cells from B6 mice throughout the study period (Fig. 2,B). T cells from other lupus-prone strains such as NZB/W F1, MRL/MpJ, and B6.MRL/lpr mice, which develop lupus pathology at a later time point, did not display increased levels of T cells with clustered lipid rafts at 10 wk of age in a manner similar to that of T cells from MRL/lpr mice (Fig. 2,C, left panel). But we observed increased levels of T cells with clustered lipid rafts in NZB/W F1 mice at the age of 8 mo and B6.MRL/lpr mice at the age of 10 mo (Fig. 2 C, right panel).
Because MRL/lpr mice have increased numbers of spontaneously activated T cells, we asked whether the observed lipid raft clustering can be reproduced by TCR stimulation. B6 mice were administered i.p. CD3 plus CD28 Abs and 16 h later the percentage of T cells displaying aggregated lipid rafts was determined. We found high levels of T cells displaying clustered lipid rafts in B6 mice following stimulation with CD3 plus CD28 Abs (Fig. 2, A and D).
Accelerated disease progression by CTB is due to enhanced lipid raft clustering
To further understand how CTB promotes disease progression in MRL/lpr mice, we performed the following experiments. First, we determined whether treatment with CTB causes lipid raft clustering on normal T cells. T cells isolated from B6 mice treated with CTB (2 μg/mouse/wk) for 4 wk were stained with FITC-CTB. CTB treatment caused lipid raft clustering in T cells in vivo (Fig. 2,E). We also found that the presence of CTB triggered lipid raft clustering in T cells from B6 mice when incubated at 37°C but not at 4°C in vitro (Fig. 2,E). Next, we determined whether CTB treatment further enhanced lipid raft clustering on T cells in MRL/lpr mice. We found that treatment of MRL/lpr mice with CTB (2 μg/wk for 4 wk) further increased the percentage of T cells with clustered lipid rafts (Fig. 2,F). In addition, we found that the numbers of T cells with clustered lipid rafts in B6 mice treated with CTB (2 μg/wk for 4 wk) and followed by the administration of CD3 Ab (70 μg/mouse 16 h before the end of the experiment) increased significantly compared with those of the control group (Fig. 2 G). Lastly, we excluded the possibility that CTB treatment caused kidney injury, as we did not observe abnormal pathology in B6 mice treated with CTB (2 μg/day i. p.) for 1 wk or weekly (2 μg i.p.) for one month (data not shown). Also, we did not observe a significant increase in the number of T cells in B6 mice treated daily with CTB (2 μg) for 1 wk compared with control mice (data not shown), excluding thus the possibility that CTB treatment accelerates disease by causing lymphoproliferation. Together, these results suggest that accelerated lupus pathology progression in MRL/lpr mice following CTB treatment is due to enhanced lipid raft clustering on T cells.
Disruption of lipid rafts delays disease development in MRL/lpr mice
We considered that if CTB enhances lipid raft clustering and advances lupus pathology, then depletion of lipid rafts should delay disease progression in MRL/lpr mice. MRL/lpr mice were treated with MβCD, which is known to disrupt lipid rafts by depleting cholesterol (9, 14, 15). We found that disease progression, as manifested by proteinuria, kidney pathology, deposition of Ig and complement in glomeruli, and serum IgG and anti-dsDNA Ab levels, was significantly reduced in MRL/lpr mice treated with MβCD compared with the PBS-treated control group at 16 wk of age (Fig. 3, A–G). We also observed that MβCD treatment (for 4 mo) extended the survival of MRL/lpr mice compared with control PBS treatment as recorded at 13 mo of age (Fig. 3 H). To confirm our results, we treated MRL/lpr mice with atorvastatin, which is known to inhibit cholesterol synthesis. We found that inhibition of cholesterol synthesis by atorvastatin also inhibited disease progression in MRL/lpr mice (our unpublished data). We did not observe obvious effect of MβCD on lymph node and spleen size in treated mice. These data provide novel proof that delayed disease progression in MRL/lpr mice by MβCD or atorvastatin is due to reduced T cell lipid raft clustering.
Delayed disease progression by MβCD is due to reduced lipid raft clustering
To understand how MβCD delayed disease progression in MRL/lpr mice, we performed the following experiments. First, we investigated whether lipid raft clustering is reduced in MRL/lpr mice treated with MβCD. We found that the percentage of T cells with aggregated lipid rafts was significantly reduced in MRL/lpr mice treated with MβCD compared with T cells from mice treated with PBS or control αCD (Fig. 4,A). Second, to determine whether MβCD inhibits TCR-mediated lipid raft clustering, B6 mice were treated with anti-CD3 Ab following the administration of MβCD for 2 wk. The results show that TCR-mediated lipid raft clustering was reduced in mice treated with MβCD compared with control-treated mice (Fig. 4 B). Third, we investigated whether MβCD treatment induces apoptosis of T cells, because delayed disease progression by MβCD might have resulted from increased T cell apoptosis. The number of T cells was similar between B6 mice treated with MβCD (1 mg) daily for 1 wk and control PBS-treated mice (data not shown). Lastly, we found that the numbers of T cells with aggregated lipid rafts was also reduced in MRL/lpr mice treated with atorvastatin (not shown). These data indicate that delayed disease progression in MRL/lpr mice by MβCD is due to reduced lipid raft clustering on T cells.
Clustered lipid rafts represent the assembly site for diverse molecules
Next, we analyzed the composition of clustered lipid rafts on T cells in MRL/lpr mice. We found that clustered lipid rafts contain T cell signaling molecules including, CD3ε, ZAP-70, Lck, PI3K, Syk, and LAT (linker for activation of T cells) (Fig. 5,A). Lipid raft clusters existed on CD4+ and CD8+ T cells in MRL/lpr mice, but not on the surface membrane of double negative CD4−CD8− T cells (Fig. 5,B). Costimulatory molecules such as CD28 and CD40L were also present in the clustered lipid rafts (Fig. 5, B and C). Inflammatory molecules such as TNF-α, TNFR2, MCP-1, and IFN-γ, which are known to play important roles in the pathogenesis of autoimmune diseases (20, 21, 22, 23), colocalized with CTB on the surface membrane of MRL/lpr T cells (Fig. 5,C). The adhesion molecule CD44, which is overexpressed in human SLE T cells (24), was found to localize in the lipid rafts of MRL/lpr T cells (Fig. 5,C). TLR molecules, including TLR4 and TLR9, were also found in lipid rafts of MRL/lpr T cells (Fig. 5,D). Finally, we found that T cells infiltrating kidney tissues expressed Syk and CD44 (Fig. 5 E). Together, these data demonstrate that clustered lipid rafts represent the assembly site for diverse signal molecules on T cells in MRL/lpr mice.
TCR costimulatory function depends on intact lipid rafts
To further understand how lipid raft clustering modulates disease progression in MRL/lpr mice, we determined whether diverse signaling molecules recruited in clustered lipid rafts contribute to TCR signaling, because lipid raft clustering may amplify and sustain TCR signaling (9). We determined whether these recruited molecules can provide costimulation to TCR-mediated T cell proliferation, as T cells from lupus-prone, Fas-intact MRL mice display increased TCR-mediated proliferation (25). We found that TCR-induced T cell proliferation was significantly enhanced in the presence of anti-CD28 Ab, anti-TNFR2 Ab, TNF-α, or MCP-1, compared with the stimulation of TCR Ab alone, and was abolished in the presence of MβCD (Fig. 6 A). These data demonstrate that molecules localized in the clustered lipid rafts have costimulatory effects on TCR signaling that depend on lipid raft clustering.
Next, we investigated whether recruited molecules promote lipid raft clustering. We determined whether CD28 promotes TCR-mediated lipid raft clustering because CD28-deficient mice are resistant to the development of lupus (26). B6 mice were treated with CD3 Ab alone or in combination with CD28 Ab. The results demonstrate that CD28 significantly enhanced lipid raft clustering on T cells in B6 mice in the presence of CD3 Ab compared with mice treated with CD3 Ab alone, and MβCD inhibited this costimulation (Fig. 6,B). These results indicate that recruited molecules further contribute to TCR-mediated lipid raft clustering. Along the same line, we investigated whether a deficiency of recruited molecules affects lipid raft clustering on T cells. Specifically, we asked whether a deficiency in CD40L reduces lipid raft clustering because CD40L-deficient are resistant to the development of lupus in MRL/lpr mice (27, 28). We found that lipid raft clustering was significantly reduced and lasted for a short time in CD40L-deficient mice treated with CD3 plus CD28 Abs compared with wild-type mice (Fig. 6 C). These data demonstrate that CD40L deficiency affected lipid raft clustering on T cells.
Additionally, we investigated whether IFN-γ production depends on lipid raft clustering, because IFN-γ is known to promote lupus pathology in MRL/lpr mice (22). We found that IFN-γ levels were markedly increased in supernatants from T cells treated with CD3 plus CD28 Abs compared with CD3 Ab alone and that increased IFN-γ levels were abolished by MβCD (Fig. 6,D). Also, serum IFN-γ levels were reduced in CD40L-deficient mice following stimulation of CD3 plus CD28 Abs compared with wild-type mice (Fig. 6 E).
Lastly, we asked whether the aggregation of lipid rafts would affect T cell function. To this end, we purified T cells from B6 mice and MRL/lpr mice (age of 9 wk) treated them with CTB and then with an anti-CTB Ab for 72 h. Control T cells were only treated with the anti-CTB Ab alone. As shown in Fig. 6 F, cross-linking of lipid rafts with CTB resulted in significantly increased IFN-γ production by both B6 (p = 0.0024) and MRL/lpr mice (p = 0.0004). It should be noted that the increase was more pronounced in the MRL/lpr T cells.
Lipid raft clustering depends on actin polymerization in MRL/lpr mice
To understand how lipid raft clustering occurs, we investigated whether actin polymerization is important for the assembly of diverse molecules (29, 30). Confocal microscopy results showed that actin colocalized with lipid rafts on T cells from MRL/lpr mice (Fig. 7,A). To further determine the role of actin in lipid raft clustering, we investigated whether the inhibition of actin polymerization limits lipid raft clustering on T cells from MRL/lpr mice. MRL/lpr mice were treated with or without cytochalasin D, an inhibitor of actin polymerization (31, 32). Cytochalasin D (1 mg/kg) was administered daily for 1 wk. We found that lipid raft clustering was significantly inhibited in MRL/lpr mice following treatment with cytochalasin D compared with control (Fig. 7 B). These results suggest that lipid raft clustering on T cells in MRL/lpr mice localizes diverse molecules through actin polymerization.
T cells with clustered lipid rafts appear in MRL/lpr mice as early as 3 wk of age and their numbers peak by the 7th wk of age. Clustered lipid rafts contain signaling, adhesion, and costimulatory molecules. Our studies demonstrate that CTB accelerates disease progression in MRL/lpr mice by enhancing the aggregation of T cell lipid rafts, whereas the disruption of lipid rafts delayed disease progression in MRL/lpr mice.
The critical role of T cells in the development of autoimmune disorders in MRL/lpr mice has been demonstrated in previous studies (33, 34, 35, 36). Enhancement of CD3-mediated signaling by costimulatory molecules facilitates T cell activation and promotes the development of autoimmunity. Clustered lipid rafts presumably provide more stable and efficient signaling platforms for TCR signaling molecules and amplify and sustain TCR signaling. Our studies demonstrate that recruited molecules, such as CD28, TNF-α, TNFR2, MCP-1, and CD40L have costimulatory activity on TCR-mediated signaling. The costimulatory function to TCR-mediated signaling depends on intact lipid rafts, because disruption of lipid rafts abolished the costimulatory effects to TCR-mediated proliferation and production of IFN-γ (Fig. 6). Cross-linked components of lipid rafts such as CD28 significantly enhanced TCR-mediated lipid raft clustering. Loss of clustered lipid raft components, such as CD40L, resulted in defective TCR-mediated lipid raft clustering. Actually, T cells that lack TNFR2 are defective in TCR-mediated activation (37). Blocking of recruited signaling molecules to lipid rafts may inhibit lupus pathology. Indeed, inhibition of PI3K has been shown to inhibit lupus pathology in MRL/lpr mice (38, 39). Lipid raft clustering may promote activated T cells extravasation from blood vessels to sites of pathological organ tissue, because components such as CD44 facilitate this process (24).
Cholera toxin (CT) from V. cholerae, a Gram-negative bacterium, may cause massive secretory diarrhea. CT is composed of an enzymatic A subunit and a homopentameric B subunit (40). CT exerts its effect through CTB, which is nontoxic and binds to GM1 (41). CTB binding to GM1 induces lipid raft clustering on mammalian cells such as T cells and epithelial cells (14, 18, 19). CTB-mediated lipid raft clustering depends on actin cytoskeleton (42). Our results demonstrate that actin polymerization plays an important role in the aggregation of lipid rafts on T cells in MRL/lpr mice. Therefore, CTB treatment may promote lipid raft clustering on T cells through actin polymerization. Clustered lipid rafts represent the assembly site of diverse signaling molecules, including TCR. TCR for specific autoantigen is also recruited to clustered lipid rafts and, therefore, autoantigens binding TCR localized in clustered lipid rafts amplify their signaling transduction and promote autoimmunity and development of autoimmune disease. Because lipid rafts also play important role in BCR signaling and B cells are critical in the pathogenesis of SLE, it is possible that CTB or MβCT treatment enhances or decreases lipid raft clustering on B cells and affects disease progression in MRL/lpr mice.
SLE patients are more prone to suffer infection-related morbidity and mortality (16). Lipid rafts from host cells are exploited by a wide range of infectious agents, including Brucella, Chlamydia, Legionella, Listeria, Pseudomonas, Salmonella, Shigella, Streptococcus, EBV, Ebola, hepatitis C, HIV, HSV and influenza viruses and some protozoa such as Toxoplasma and Plasmodium to enter, survive, and replicate within the cell (5, 7). By activating G proteins, bacterial pathogens use the cytoskeleton to invade a host cell and to gain motility in the cell. G protein activation, in turn, induces the generation of actin-rich membrane ruffles to internalize the bacteria (43). G proteins and actin cytoskeleton are involved in T cell activation and TCR signaling (44, 45). Other studies and our study demonstrate that TLRs, including TLR4, 7, and 9 (46, 47, 48), are localized to lipid rafts. TLR binding to pathogen-associated molecular patterns may promote lipid raft clustering that, in turn, provides a stable platform to amplify TLR signaling transduction. TLR-mediated cell signaling also depends on intact lipid rafts (46, 47, 48).
Poorly understood interactions between environmental and genetic factors lead to the development of autoimmune diseases. Genetic factors may account for the increased production of molecules, including CD3-associated signaling molecules and TLRs, whereas components of infectious agents provoke the appearance of SLE by enhancing lipid raft clustering. Patients treated with immunosuppressive agents such as prednisone and cytotoxic drugs become prone to suffer infections that, in turn, may cause disease reactivation. Our results demonstrate that lipid raft clustering contributes to the pathogenesis of SLE, because its dissolution delays disease whereas acceleration of its formation promotes disease pathology. Clustered lipid rafts in MRL/lpr mice T cells harbor various molecules that appear to amplify and maintain TCR-mediated signaling. Our data strongly suggest that lipid rafts provide a potential link though which infections instigate or promote autoimmune disease.
We thank Dr. P. Lapchak for critically reading the manuscript.
We have no financial conflict of interest.
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.
This work was supported in part by National Institutes of Health Grant R01AI42269.
Abbreviations used in this paper: SLE, systemic lupus erythematosus; B6, C57BL/6 mice; αCD, α-cyclodextrin; C3, complement 3; CD40L, CD40 ligand; CT, cholera toxin; CTB, cholera toxin B; MβCD, methyl-β-cyclodextrin; NZB/W F1, (New Zealand Black × New Zealand White)F1 mice; PAS, periodic acid-Schiff.