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Spinster 2, a sphingosine-1-phosphate transporter, plays a critical role in inflammatory and autoimmune diseases.

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Affiliations

  • 1. *Lexicon Pharmaceuticals Incorporated, The Woodlands, Texas, USA; and Department of Biochemistry and Molecular Biology, and Department of Surgery and Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA.
      Authors
    • Donoviel MS 1
    • Hait NC 1
    • Ramachandran S 1
    • Maceyka M 1
    • Takabe K 1
    • Milstien S 1
    • Oravecz T 1
    (7 authors)
  • 2. *Lexicon Pharmaceuticals Incorporated, The Woodlands, Texas, USA; and Department of Biochemistry and Molecular Biology, and Department of Surgery and Massey Cancer Center, Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA
      Authors
    • Spiegel S 2
    (1 author)

ORCIDs linked to this article

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology, 31 Aug 2015, 29(12):5018-5028
https://doi.org/10.1096/fj.15-274936 PMID: 26324848 PMCID: PMC4653056

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Abstract 

Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive sphingolipid metabolite that regulates numerous processes important for immune responses. S1P is made within cells and must be transported out of cells to exert its effects through activation of 5 specific cell surface GPCRs in an autocrine or paracrine fashion. Spinster 2 (Spns2) transports S1P out of cells, and its deletion in mice reduces circulating levels of S1P, alters immune cell trafficking, and induces lymphopenia. Here we examined the effects of Spns2 deletion on adaptive immune responses and in autoimmune disease models. Airway inflammation and hypersensitivity as well as delayed-type contact hypersensitivity were attenuated in Spns2(-/-) mice. Similarly, Spns2 deletion reduced dextran sodium sulfate- and oxazolone-induced colitis. Intriguingly, Spns2(-/-) mice were protected from the development of experimental autoimmune encephalopathy, a model of the autoimmune disease multiple sclerosis. Deletion of Spns2 also strongly alleviated disease development in collagen-induced arthritis. These results point to a broad role for Spns2-mediated S1P transport in the initiation and development of adaptive immune related disorders.

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FASEB J. 2015 Dec; 29(12): 5018–5028.
Published online 2015 Aug 31. https://doi.org/10.1096/fj.15-274936
PMCID: PMC4653056
PMID: 26324848

Spinster 2, a sphingosine-1-phosphate transporter, plays a critical role in inflammatory and autoimmune diseases

Michael S. Donoviel,*,1 Nitai C. Hait,† Subramaniam Ramachandran,† Michael Maceyka,† Kazuaki Takabe,†‡ Sheldon Milstien,† Tamas Oravecz,*,2,3 and Sarah Spiegel†,3,4
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Abstract

Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive sphingolipid metabolite that regulates numerous processes important for immune responses. S1P is made within cells and must be transported out of cells to exert its effects through activation of 5 specific cell surface GPCRs in an autocrine or paracrine fashion. Spinster 2 (Spns2) transports S1P out of cells, and its deletion in mice reduces circulating levels of S1P, alters immune cell trafficking, and induces lymphopenia. Here we examined the effects of Spns2 deletion on adaptive immune responses and in autoimmune disease models. Airway inflammation and hypersensitivity as well as delayed-type contact hypersensitivity were attenuated in Spns2−/− mice. Similarly, Spns2 deletion reduced dextran sodium sulfate– and oxazolone-induced colitis. Intriguingly, Spns2−/− mice were protected from the development of experimental autoimmune encephalopathy, a model of the autoimmune disease multiple sclerosis. Deletion of Spns2 also strongly alleviated disease development in collagen-induced arthritis. These results point to a broad role for Spns2-mediated S1P transport in the initiation and development of adaptive immune related disorders.—Donoviel, M. S., Hait, N. C., Ramachandran, S., Maceyka, M., Takabe, K., Milstien, S., Oravecz, T., Spiegel, S. Spinster 2, a sphingosine-1-phosphate transporter, plays a critical role in inflammatory and autoimmune diseases.

Keywords: export, lymphopenia, innate immunity, adaptive immunity

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite that exerts many of its effects through the activation of 5 cell surface GPCRs (S1PR1–5). Signaling downstream of these receptors is involved in many physiologic and pathophysiologic responses important for innate and adaptive immunity, including immunosurveillance, immune cell trafficking and differentiation, immune responses, and endothelial barrier integrity (1). S1P levels are in the micromolar range in mammalian blood, lower in lymph, and it is thought that an S1P gradient exists among blood, lymph, and tissues that controls trafficking of immune cells between the circulation and lymphoid tissues (2). Clinically, the prodrug FTY720 (fingolimod), which is phosphorylated to FTY720-P, a S1P mimetic, is used in the treatment of multiple sclerosis. FTY720-P promotes the internalization and degradation of S1PR1 and induces lymphopenia by preventing the egress of T and B cells from secondary lymphoid organs to sites of inflammation (3).

S1P is produced in cells by phosphorylation of sphingosine by 2 sphingosine kinases, SphK1 and SphK2. Thus, mechanisms must exist for cells to export S1P to the extracellular milieu. Indeed, several plasma membrane transporters of S1P have been identified, including ABC transporter family members and the major facilitator superfamily member, Spinster 2 (Spns2) (reviewed in refs. 4, 5). A role for Spns2 in S1P secretion was first suggested by experiments in zebrafish, where it was demonstrated that Spns2 mutation induced a similar cardiac defect as an S1P receptor mutant (6, 7). Moreover, this phenotype could be rescued by exogenous addition of S1P (7). Intriguingly, mice genetically deleted of functional Spns2 protein develop and reproduce normally, though having altered lymphocyte trafficking (8–12) and early-onset hearing loss (13). Several in vitro studies have convincingly demonstrated that Spns2 can export S1P as well as S1P analogs (7, 8, 12, 14).

We examined the contribution of Spns2 to regulation of innate immune and inflammatory responses in mice. Our studies demonstrated that Spns2 plays an important role in the control of allergen-induced asthma, delayed-type hyperresponsiveness, and colitis and in the initiation and progression of autoimmunity in mouse models of multiple sclerosis and rheumatoid arthritis. Our results, taken together, suggest that Spns2 is an attractive drug target for the treatment of these diseases.

MATERIALS AND METHODS

Mice

Spns2−/− mice were generated by homologous recombination replacing exon 3 of the Spns2 gene (GenBank accession no. AB441166) with a β-Geo/puro selection cassette (Supplemental Fig. 1A). Southern blot hybridization analysis demonstrated the targeted mutation in embryonic stem cells (Supplemental Fig. 1B). Heterozygote (Spns2+/−) mice were viable and fertile. Mating of Spns2+/− mice generated pups of the 3 possible genotypes with ratios that fit well within normal (1:2:1) mendelian frequencies [25% wild-type (WT, Spns2+/+), 51% Spns2+/−, 24% Spns2−/−; n = 2028; χ2 = 1.28, P = 0.55]. This result indicated that Spns2 is not essential for neonatal viability. The body weight of male Spns2−/− mice was 11.4% lower on average than that of WT littermates throughout their adulthood, although weight differences in female mice were not significantly different (Supplemental Fig. 1C).

All mice analyzed were maintained on a mixed genetic background (129S5/SvEvBrd and C57BL/6J) at the Association for Assessment and Accreditation of Laboratory Animal Care–accredited animal facility at Lexicon Pharmaceuticals, Inc. (The Woodlands, TX, USA). Heterozygote (Spns2+/−) mice were always used to generate Spns2−/− mice, and studies of gene-disrupted animals were performed using WT littermates as controls. Mice were housed in a barrier facility at 22°C on a fixed 12 h light and 12 h dark cycle and were fed with mouse chow and water ad libitum. Procedures involving animals at Lexicon and Virginia Commonwealth University were conducted in conformity with the Institutional Animal Care and Use Committee guidelines that are in compliance with the state and federal laws and the standards outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA).

Complete blood count and flow cytometry

Complete blood count (CBC) analysis was performed on blood collected from the retro-orbital sinus of mice anesthetized with isoflurane as previously described (15). Flow cytometry analysis of lymphocyte subsets in whole blood and in single cell suspensions from thymus, spleen, and lymph nodes was performed with a FACSCalibur flow cytometer with CellQuest software (Becton Dickinson, San Diego, CA, USA) after staining with different combinations of fluorochrome-coupled antibodies as described (15).

β-Galactosidase staining and LacZ histochemistry

Tissue sections were fixed and stained for β-galactosidase (β-Gal) activity essentially as described (16). Briefly, anesthetized mice were perfused sequentially via the left ventricle with 8 ml of saline solution; then 10 ml of β-Gal fixative (0.2% glutaraldehyde, 1.5% paraformaldehyde, 2 mM MgCl2, 5 mM ethylene glycol, 100 mM sodium phosphate, pH 7.3), followed by 2 ml of β-Gal rinse [0.2% Nonidet-P40 (NP-40), 0.1% sodium deoxycholate, 2 mM MgCl2, 100 mM sodium phosphate] and finally 10 ml of β-Gal stain [5 mM K3Fe(CN)6 (potassium ferricyanide), 5 mM K4Fe(CN)6 (potassium ferrocyanide), 1 mg of 5-bromo-4-chloro-3-indolyl-d-galactopyranoside (β-Gal) (dissolved in dimethylformamide) per milliliter, 0.2% NP-40, 0.1% sodium deoxycholate, 2 mM MgCl2, 100 mM sodium phosphate, pH 7.3]. Tissues were then dissected and postfixed in a β-Gal fix for 20 min, rinsed 3 times for 10 min in the β-Gal rinse, and then incubated in the β-Gal stain solution for 48 h. After 3 additional 10 min washes in β-Gal rinse, the tissues were placed in Bouin fixative for 24 h before dehydration and embedding in paraffin. Sections were cut at 4 μm and counterstained with nuclear fast red.

Induction of allergic inflammation and airway hyperresponsiveness

Sensitization with ovalbumin (OVA) and alum and then intranasal challenge with OVA were performed as previously described (17). Bronchiolar lavage (BAL) fluid was collected by lavaging the lungs. Cells and supernatants were collected by centrifugation, and proportions of different cell types quantified. IL-4, IL-5, and IL-13 were determined by cytometric bead array–based mouse cytokine Flex Sets (BD Biosciences, San Jose, CA, USA) (18). Levels of OVA-specific IgG1 and IgG2a were measured by OVA-specific ELISA (Cayman Chemicals, Ann Arbor, MI, USA) according to the manufacturer’s protocol (18). In some experiments, mice were anesthetized and after tracheotomies were ventilated, and measurements of baseline lung function were made with the FlexiVent apparatus (Scireq, Montreal, QC, Canada) as described (19). Mice were then exposed to aerosols containing acetyl-β-methylcholine chloride and lung resistance (RL),measured by FlexiVent 5.3 (19).

Contact hypersensitivity assay

Mice were sensitized to oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-1; Sigma-Aldrich, St. Louis, MO, USA) by epicutaneous application of 5% oxazolone to shaved portions of their abdomen. Mice were then rechallenged with oxazolone 6 d later. For the delayed-contact hypersensitivity assay, one ear of each mouse was painted with 10 µl of 3% oxazolone on each side, while the other ear was painted with vehicle. The changes in ear swelling were then compared over 72 h for each mouse with a Käfer thickness micrometer (20).

Induction and analysis of acute colitis

Acute colitis was induced with 5% dextran sulfate sodium (DSS) in drinking water for 7 d, followed by 7 d of normal water. Mice were weighed daily to assess weight loss. Histologic assessments of colitis and severity scores were made in a double-blinded manner after hematoxylin and eosin (H&E) staining (21, 22). Oxazolone colitis was induced as previously described (21). Briefly, mice were sensitized by topically applying 3% oxazolone on the shaved abdomen. Seven days later, 1% oxazolone was administered intrarectally.

Experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) was induced as previously described (18). Briefly, Spns2−/− and WT littermates were injected subcutaneously with 300 µg myelin oligodendrocyte glycoprotein (MOG) p35–55 emulsified in complete Freund adjuvant containing 250 µg heat-inactivated Mycobacterium tuberculosis H37Ra (BD Biosciences), immediately followed by intravenous injection of 500 ng of Pertussis toxin (List Biologic Laboratories, Campbell, CA, USA). Disease severity was scored over 3 wk on a scale of 0 to 5 as previously described (23). The percentage of mice exhibiting any EAE symptoms was also scored. Histopathologic analysis was performed essentially as described (18). Briefly, fixed, paraffin-embedded sections of brain (3 to 5 sections) were stained with H&E and scored for inflammatory and degenerative lesions using the following scale: 0 = absent, 1 = minimal, 2 = conspicuous focal, 3 = prominent focal, 4 = marked multifocal, 5 = severe diffuse.

Collagen-induced arthritis and collagen antibody–induced arthritis

The induction of collagen-induced arthritis was performed essentially as described (24). Briefly, collagen type II and M. tuberculosis were emulsified in incomplete Freund adjuvant and injected intradermally at the base of the tail at d 0 and 21, and mice were monitored for an additional 40 d for signs of arthritis (24). Collagen antibody–induced arthritis was induced by the Arthrogen-CIA Arthritogenic 5-clone Monoclonal Antibody cocktail kit (catalog #53100; Chondrex, Redmond, WA, USA), according to the manufacturer’s instructions. Disease severity in both models was assessed visually on a scale of 0 to 4 of increasing swelling and erythema and the extent of involvement of ankles, feet, and digits. The arthritis score is the sum of these scores for all 4 limbs for all mice involved (n = 21 for each genotype). A Käfer thickness micrometer was used to measure the forelimb ankle thickness, with thickness averaged over the 2 forelimbs, and the data expressed as the change in thickness for the mice over the course of the experiment.

Mass spectrometry

Sphingolipids from tissues were extracted and quantified by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) (4000 QTRAP; Applied Biosystems, Foster City, CA, USA) as described (25).

Immunoblotting

Tissue samples were homogenized in buffer containing 50 mM Tris (pH 7.4), 100 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM DTT, and 0.2 mM PMSF, with a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). Proteins were resolved by SDS-PAGE and blotted to nitrocellulose. After blocking, Spns2 protein was visualized with Spns2-specific antibody (#SAB2104271-50UG, 1:1000 dilution; Sigma-Aldrich) and appropriate horseradish peroxidase–conjugated secondary (1:5000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) followed by enhanced chemiluminescence. Blots were stripped and reprobed with tubulin antibody (Cell Signaling Technology, Danvers, MA, USA) to confirm equal protein loading.

Statistical analysis

Statistical analysis was performed by unpaired 2-tailed Student’s t test for comparison of 2 groups and ANOVA with Bonferroni post hoc comparison for experiments consisting of 3 or more groups. P < 0.05 was considered significant. Statistical differences in mean daily clinical scores and histology scores in mice with EAE or collagen-induced arthritis were analyzed by the nonparametric Mann-Whitney U test or Kruskal-Wallis test with Dunnett’s multiple-comparison post hoc analysis.

RESULTS

Spns2−/− mice are lymphopenic

Although several studies have demonstrated that S1P levels in blood are reduced in Spns2−/− mice (8, 9, 12), others have reported that plasma S1P levels are not reduced in these mice (10, 11). We found that levels of S1P were significantly lower in platelet-poor plasma and in blood from Spns2−/− mice compared with WT littermates (Fig. 1A). Given the role of circulating S1P in immune cell trafficking (2), and in agreement with others (8–12), CBC showed that lymphocyte numbers were greatly decreased in Spns2−/− mice compared with WT littermates, although numbers of eosinophils, basophils, neutrophils, and monocytes, as well as erythrocytes and platelets, were not significantly different (Fig. 1B). Also, other red blood cell indices were not significantly affected by deletion of Spns2 (Supplemental Table 1). Furthermore, Spns2-knockout mice have a strong reduction in circulating CD4 and CD8 single positive (SP) T cells, and B cells showed a similar trend, although less dramatic (Fig. 1C), as reported previously (11). CD4+ and CD8+ T cells were also lower in secondary lymph organs, spleen, and lymph nodes from Spns2−/− mice (Fig. 1D,E) and conversely were increased in thymus (Fig. 1F). Further analysis of the SP populations revealed that the proportion and number of CD62LhiCD69lo CD4 SP and CD8 SP thymocytes were increased in Spns2-deficient mice (Fig. 1G). These phenotypic changes in the Spns2−/− mature thymocyte population are consistent with a block in the migration of lymphocytes into the periphery and a concomitant accumulation of mature thymocytes, described as recent thymic emigrants in the thymus of the Spns2-deficient animals. Together, these results are consistent with the notion that Spns2 is important for S1P-dependent lymphocyte trafficking.

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Spns2 deletion decreases blood S1P levels and affects lymphocyte trafficking. A) Spns2−/− mice and WT littermates were euthanized and S1P levels in whole-blood, platelet-poor plasma (PPP) measured by LC-ESI-MS/MS. Data are expressed as picomoles per milliliter and are means ± sd, *P < 0.05. B–G) Hematopoietic cell profile of blood and indicated tissues from WT (open bars, n = 19) and Spns2−/− (solid bars, n = 22) mice determined by CBC (B) and FACS analysis (C–F). Further FACS analysis was performed on CD4 and CD8 SP thymocytes (G) gated for maturation markers CD69 and CD62L to determine number of CD62Lhi CD69lo T cells. Data are presented as means ± sem, *P < 0.01. PTL, platelets; RBC, red blood cell; lymph, lymphocytes; mono, monocytes; neutro, neutrophil granulocytes.

These results raise the question of whether the effects on lymphocyte trafficking are caused solely by decreased circulating levels of S1P in Spns2-knockout mice. However, this seems unlikely. Although no differences were noted in the percentages of T cells and B cells in blood or lymphoid organs in SphK1−/− mice (26), similar to Spns2−/− mice, they have significant reductions in blood S1P compared with WT littermates (Fig. 2). Moreover, whereas increases in S1P were found in thymus, liver, and colon from Spns2−/− mice that were saline perfused to remove circulating S1P, in SphK1−/− mice, in contrast, S1P levels in these organs were significantly decreased (Fig. 2).

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Comparison of S1P levels in tissues from SphK1−/− and Spns2−/− mice and their littermates. Tissues were isolated from saline-perfused SphK1−/− and Spns2−/− mice and their respective WT littermates (n = 4). Lipids were extracted from blood and tissues, and S1P was determined by LC-ESI-MS/MS. Data are presented as means ± sd, *P < 0.05, compared with WT.

Redistribution of lymphocytes in mice lacking Spns2 is not dependent on hematopoietic cells

Taken together, our results suggest that there is an aberrant S1P gradient between the circulation and tissues in Spns2−/− mice, which leads to a defect in lymphocyte trafficking and lymphopenia. To substantiate this, Spns2−/− and WT mice were used to generate reciprocal groups of bone marrow (BM) chimeras by transplanting Spns2−/− BM into lethally irradiated WT hosts and vice versa. Peripheral blood was subsequently analyzed in both fully reconstituted cohorts. When WT mice were analyzed 20 wk after transplantation with Spns2−/− BM, they did not exhibit an abnormal blood cell profile (Fig. 3). On the other hand, Spns2−/− mice reconstituted with WT BM showed a blood phenotype similar to that seen in naive Spns2−/− mice (Fig. 3); that is, WT hematopoietic cells could not rescue the blockage of T-cell egress noted in Spns2−/− mice. Data from these studies support the notion that expression of Spns2 in nonhematopoietic cell types, presumably in the primary and secondary lymphoid organs themselves, is critical for normal immune cell migration (9, 10).

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BM chimeras. Flow cytometric analyses of WT and Spns2−/− mice that were reconstituted with littermate BM (BM chimera) from WT or Spns2−/− mice as indicated. Data are presented as means ± sem, *P < 0.01, compared with WT donor BM > WT recipient.

The IRES/LacZ motif within the selection cassette used to disrupt Spns2 created a β-Gal fusion transcript that recapitulated the expression pattern of the endogenous gene and allowed β-Gal expression in Spns2−/− mice to be used as a surrogate marker. In agreement with Hisano et al. (8), β-Gal staining was detected in endothelial cells in the thymus (Supplemental Fig. 2). There was intense β-Gal staining in the white pulp of the spleen in reticular cells delimiting the marginal zone separating the white pulp from the red pulp (Supplemental Fig. 2). In lymph node, prominent staining of reticular cells lining medullary chords and high endothelial venules was noted. These results indicate that Spns2 expression in nonlymphoid cells is localized to regions of lymphatic tissues associated with lymphocyte egress.

Airway inflammation and delayed-type contact hypersensitivity are attenuated in Spns2−/− mice

Surprisingly, although the systemic redistribution of lymphocytes in Spns2-knockout mice has been reported in several studies (8–12), effects on adaptive immune responses, and particularly in autoimmune disease models, have not been investigated; it was only reported that antibody responses to immunization are reduced in Spns2-knockout mice (10). Therefore, it was important to examine whether the effects of Spns2 deletion on lymphocyte circulation also impaired immune responses.

In OVA-induced allergic asthma, as expected, WT mice developed symptoms of airway inflammation with an influx of eosinophils (Fig. 4A), the main drivers of allergic asthma, elevated Th2 cytokine levels (Fig. 4B), and production of OVA-specific IgG (Fig. 4C) and airway hyperresponsiveness (Fig. 4D). In OVA-challenged Spns2−/− mice, eosinophils and lymphocytes in BAL fluid were decreased compared with WT littermates, whereas macrophage numbers were increased. Cytokines, including TH2-type IL-4 and IL-13, which have been implicated in the induction of airway hyperresponsiveness, and IL-5, the primary chemotactic factor in the recruitment of eosinophils, were all also significantly reduced in Spns2−/−-challenged mice (Fig. 4B). Spns2-knockout mice also produced lower amounts of OVA-specific serum IgG1 after challenge compared with their WT littermates (Fig. 4C). Furthermore, Spns2−/− mice had significantly less methacholine-induced airway resistance compared with that seen in WT mice (Fig. 4D).

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Spns2−/− mice have attenuated airway inflammation and hyperresponsiveness. A) WT (n = 20) and Spns2−/− (n = 18) mice were sensitized and challenged with OVA. BAL fluid was collected and numbers of eosinophils, neutrophils, macrophages, and lymphocytes were determined. B) Levels of indicated cytokines and chemokines in BAL fluid were measured by ELISA. C) OVA-specific IgG1 were measured by OVA-specific ELISA. D) Separate groups of WT and Spns2−/− (n = 6) mice were sensitized and challenged with OVA. Airway responses to methacholine were measured with FlexiVent apparatus 24 h after last OVA challenge. Data shown are lung resistance ΔRL (cmH2O · s · ml−1). *P < 0.01 compared to OVA-challenged WT mice. E) Oxazolone-induced contact hypersensitivity is reduced in Spns2−/− mice. WT (n = 31) and Spns2−/− (n = 24) mice were sensitized to oxazolone and challenged on right ear 6 d later. Changes in ear swelling were determined at indicated times. Data are presented as means ± sem, *P < 0.01, compared with WT.

To study the role of Spns2 in delayed-type hypersensitivity, we used a common mouse model of allergic contact dermatitis. Mice were sensitized to oxazolone epicutaneously, and delayed-contact hypersensitivity was induced in one ear by reexposure 6 d later. Sensitization of WT mice with oxazolone induced antigen-specific swelling as a readout of inflammation-induced edema formation that peaked at 24 h after the challenge and remained elevated for at least 3 d. In contrast, Spns2−/− mice showed impaired ear-swelling responses compared with WT mice (Fig. 4E).

Spns2 deletion reduced chemically induced intestinal inflammation

Because S1P has been implicated in the development of DSS-induced colitis, and deletion of SphK1, which decreases circulating levels of S1P, reduces colitis severity (27), we wondered whether Spns2 deletion, which also decreases circulating S1P, reduces colitis severity. To this end, 2 murine models of intestinal inflammation were utilized. In the DSS model, mice were given drinking water supplemented with DSS, which is toxic to colonic epithelial cells of the basal crypts, to induce acute colitis. Spns2-knockout mice showed less severe colitis, with significantly less weight loss than WT littermates (Fig. 5A). Histopathologic analysis also revealed less severely damaged colonic mucosa with minor loss of crypt structures and epithelial cell denudation (Fig. 5B), which was also reflected in the pathologic assessment of colitis severity scores (Fig. 5C). Moreover, whereas DSS induced significant increases in blood S1P levels in WT mice, as reported previously (22, 27), blood S1P was not increased in DSS-treated Spns2−/− mice (Fig. 5D). Interestingly, DSS did not increase Spns2 expression in the colon (Fig. 5E). Moreover, expression of other known ABC transporters of S1P (Fig. 5E) was not up-regulated to compensate for a lack of Spns2.

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Colitis is attenuated in Spns2−/− mice. A–C) Acute colitis was induced in Spns2−/− mice (n = 17) and WT (n = 19) littermates with 5% DSS in drinking water followed by recovery on normal drinking water. Changes in body weight (A), mucosal histology examined by H&E staining (B), and colitis severity scores (C) were determined in double-blind manner. Shown are number of mice with cumulative histopathology scores <3 and >3. D) Blood levels of S1P were determined by LC-ESI-MS/MS (n = 3). Data are presented as means ± SD, *P < 0.05 compared with WT. E) Proteins in colon extracts were separated by SDS-PAGE and immunoblotted with indicated antibodies. F) Spns2−/− (n = 28) and WT (n = 30) mice were administered oxazolone rectally after skin sensitization, and colitis severity was assessed by weight loss. Data are presented as means ± sem, *P < 0.001 compared with WT.

Similar results were observed when colitis was induced by intrarectal administration of oxazolone in sensitized mice, which induces a T-cell–mediated response (21). Spns2−/− mice recovered more quickly from colitis, as evidenced by more rapid weight gain between d 2 and 7, with complete weight recovery by d 8, compared with WT littermates (Fig. 5F). These results suggest that Spns2 deletion is protective in innate immune mechanisms of colitis.

Spns2 ablation protects against EAE

We next compared the responses of Spns2−/− mice with WT littermates in monophasic EAE, a widely used mouse model of multiple sclerosis. Autoimmune demyelination was induced by injection of MOG and adjuvant (18). As expected, WT mice developed EAE, which followed a typical disease course, starting at d 12 after immunization and reaching a mean maximal clinical score of 3.5 (Fig. 6A). While ~90% of the WT mice developed symptoms of EAE within 2 wk, less than 20% of the Spns2-knockout mice responded (Fig. 6B). Moreover, the severity of disease in the knockout mice was dramatically reduced. Spns2-knockout mice had reduced clinical symptoms of disease over the course of the experiment (Fig. 6A–C), and there was a 10-fold decrease in the mean cumulative disease scores in Spns2−/− mice compared with their WT littermates (Fig. 6C). In line with the findings that Spns2−/− mice are protected from EAE, histopathologic staining of cerebellum showed that the intense perivascular cuffs of mononuclear cells, a hallmark of EAE and multiple sclerosis, were essentially absent in Spns2−/− mice (Fig. 6D). Taken together, these results suggest that lymphopenia and/or decreased inflammatory cell infiltration in Spns2−/− mice may be responsible for their resistance to induction of EAE.

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Spns2−/− mice are resistant to experimental autoimmune encephalopathy. EAE was induced in WT and Spns2−/− mice (n = 18) by immunization with MOG and adjuvant and scored for disease severity over 20 d. Average clinical severity scores (A) and EAE disease incidence, percentage of mice exhibiting EAE symptoms (B) were determined. C) Representative H&E-stained sections of cerebellum of WT and Spns2−/− mice obtained on d 21 after immunization. Images depict increased inflammation in WT mice, including perivascular cuffing by mononuclear cells. D) Cumulative histopathology scores from 5 H&E-stained brain slices scored for inflammatory and degenerative lesions. Data are presented as means ± sem, *P < 0.008 compared with WT. Bar sizes are indicated.

Deletion of Spns2 alleviates disease development in collagen-induced arthritis

To further examine the role of Spns2 in autoimmune diseases, we examined the development of collagen-induced arthritis as a surrogate model of human rheumatoid arthritis (28), a systemic inflammatory disorder that primarily affects joints. WT mice developed the first clinical signs of arthritis by 25 d after immunization with type II collagen (Fig. 7A), with increasing ankle swelling (thickness), while the Spns2−/− mice did not exhibit significant swelling until 20 d later (Fig. 7B), and it was much less severe than in the WT littermates (Fig. 7A, B). Histopathologic analysis of the ankles from these mice demonstrated that WT mice had increased arthritis severity with thicker synovia and increased inflammatory infiltration and osteolysis (Fig. 7C), whereas in contrast, the Spns2−/− mice had inconspicuous, thin synovium. Similarly, the Spns2−/− mice had far less severe disease as measured by the arthritis severity score (Fig. 7D). Spns2 deletion also reduced the serum levels of MMP-3, a matrix metalloproteinase that degrades collagen and is thought to play a significant role in the pathogenesis of rheumatoid arthritis (Fig. 7E). We also observed that Spns2 deletion reduced the circulating levels of anti-collagen antibodies (Fig. 7E), suggesting that Spns2 deletion also reduced production of autoantibodies. Nevertheless, Spns2−/− mice also had reduced symptoms in collagen antibody-induced (passive) arthritis (Supplemental Fig. 3). The latter finding indicates that prevention of arthritis development by Spns2 deficiency does not necessarily require inhibition of the antigen-specific branch of the immune response. This finding highlights the role of Spns2 in regulating nonlymphoid inflammatory response or responses, in addition to lymphocyte migration and autoantibody production

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Spns2−/− mice are resistant to collagen-induced arthritis. Spns2−/− mice (n = 10) and WT littermates (n = 21) were injected with collagen and boosted with another injection 21 d later. Starting at d 25, mouse forelimbs were scored for arthritis severity (A), and swelling of both ankles was measured with calipers (B). C–E) On d 60, mice were euthanized and blood drawn by cardiac puncture. C) Representative H&E-stained sections of forelimb ankles. Arrows indicate thick synovium, inflammation, osteolysis (WT) and inconspicuous, thin synovium (Spns2−/−). D) Number of mice with cumulative histopathology scores <3 and >3. E) Serum levels of MMP-3 and anti–collagen II antibodies were measured by ELISA.

DISCUSSION

Spns2 belongs to the solute carrier family 22 (SLC22), a large family of organic ion transporters within the major facilitator superfamily (29, 30). It was previously convincingly demonstrated that Spns2 exports S1P from cells, which then can activate the S1PRs present on the cell surface (reviewed in refs. 4, 5). Results with Spns2-deficient cells showed that S1P release from endothelial cells, but not from platelets or erythrocytes, was decreased (8). Moreover, we, in agreement with several other reports (8, 9, 11, 12), have shown that plasma S1P is significantly reduced, although to a much lesser extent than was previously reported for erythrocyte-specific disruption of SphK1 (31), implying that Spns2 is not the sole determinant of plasma S1P concentration. In addition, we and others have shown that Spns2-knockout mice have decreased numbers of circulating CD4 and CD8 T cells and accumulate mature T cells in thymus. Mature recirculating B cells were also reduced in the BM from these mice as well as in blood and secondary lymphoid organs (8, 9, 11, 12). However, the reduction in blood levels of S1P in Spns2-knockout mice cannot by itself explain aberrant lymphocyte trafficking because SphK1-knockout mice have an even greater reduction in blood S1P levels but do not have lymphopenia (32). Moreover, different results between global knockout of Spns2 and endothelial-specific knockout of Spns2 (11, 12) also suggest that Spns2 in nonendothelial cells could contribute to regulation of S1P. Indeed, we observed that levels of S1P in tissues such as thymus, liver, colon, mesenteric lymph node, and lung (12) where Spns2 is expressed (10) are increased in Spns2-knockout mice. In contrast, in the global SphK1-knockout, levels of S1P in these tissues are greatly reduced. This result suggests that in Spns2−/− mice, but not in SphK1−/− mice, there is also an aberrant S1P gradient. Because the physiologic high S1P concentration gradient between the blood and secondary lymphoid tissues is an essential factor for lymphocyte egress (2, 31), it seems likely that the aberrant gradient leads to a defect in lymphocyte trafficking and lymphopenia. It is tempting to speculate that increased S1P found in lymphoid tissues of the Spns2-knockout mice could down-regulate S1PR1 expression on the lymphocytes required for their egress from the lymph node.

In several murine models of adaptive immune responses, we observed that deletion of Spns2 reduced the severity of the diseases consistent with the role of Spns2 in lymphocyte circulation. In mice sensitized to OVA, Spns2 deficiency suppressed the development of airway hyperresponsiveness and chronic airway inflammation, including infiltration of eosinophils and antigen-specific antibody production. The reduction of IL-4, IL-5, and IL-13 in these mice suggests that Spns2 deletion decreases the Th2-dominated inflammatory response in the airway, an integral component in the pathogenesis of allergic asthma. Spns2-knockout mice also had significantly reduced inflammatory responses to oxazolone in the skin compared with WT mice. Because the oxazolone-induced acute allergic contact dermatitis mouse model that we used primarily exhibits Th1 responses (33), our results suggest a role of Spns2 also in regulation of Th1-dominated inflammatory skin diseases. Moreover, Spns2 deletion also reduced disease severity in 2 well-established animal models of chronic inflammatory bowel disease, which is a prominent feature of ulcerative colitis and Crohn’s disease. The effect of Spns2 in oxazolone-induced colitis highlights its importance in Th2-dependent responses to intestinal inflammation. However, as T- and B-cell–deficient scid or Rag1−/− mice also develop severe DSS-induced intestinal inflammation (34), our results imply that Spns2 also contributes to regulation of innate immune mechanisms of colitis. Hence, Spns2 deletion was protective in both Th1- and Th2-dependent models of inflammation, suggesting that Spns2 plays a broad role in the development of the adaptive immune response.

Most impressively, Spns2 ablation almost completely prevented development of autoimmune diseases in mice, including collagen-induced arthritis and EAE. These in vivo observations underscore the essential role of Spns2 in T-cell–mediated adaptive in vivo responses to self-antigens in autoimmune diseases. Additionally, we observed that Spns2−/− mice had reduced symptoms in another model of arthritis induced by injection of anti-collagen antibodies, indicative of its involvement also downstream of the development of autoantibodies. Taken together, these data suggest that Spns2 deletion protects against both the initiation and progression of inflammatory disease. Previous studies have suggested that S1P is increased in synovial fluid of rheumatoid arthritis patients (35) and in the lumbar spinal cord of EAE mice (36). Thus, it is possible that in addition to affecting lymphocyte trafficking, Spns2 also prevents local elevations of S1P in these disease settings. Intriguingly, mice deleted for SphK1 also have reduced levels of blood S1P that are similar to those observed in Spns2−/− mice with less severe disease in both DSS-induced colitis (27) as well as OVA-induced airway hyperresponsiveness (37), although the role of SphK1 in EAE is unknown. Although SphK1-deficient mice are protected against development of TNF-α–induced arthritis (38), SphK1 deletion is not protective in the collagen-induced arthritis model (39). Furthermore, SphK1 deletion does not induce lymphopenia (26). Therefore, while alteration of lymphocyte trafficking likely plays a significant role in immunosuppression in Spns2-knockout mice, changes in S1P homeostasis and local S1P gradients may also affect other biologic processes, contributing to the potent anti-inflammatory effects. These processes may include the inflammatory responses of cell types as diverse as endothelial cells, mast cells, macrophages, and NK cells whose numbers in circulation are not altered by deletion of Spns2. However, it is also possible that in the absence of Spns2 during development, adaptive mechanisms could have been engaged that might also contribute to the resilience to inflammatory insults. Finally, the findings we describe here should promote a search for novel compounds that are potent inhibitors of Spns2 transporter activity with desirable pharmacokinetic properties that might be useful, particularly for treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.

Acknowledgments

The authors acknowledge A. M. Digeorge-Foushee, K. Millerchip, and J. Hazelwood for their help in some of the experiments. The authors also thank the Pathology Department of Lexicon Pharmaceuticals for help in preparing slides from tissue sections. This study was supported by U.S. National Institutes of Health (NIH) National Institute of General Medical Sciences Grant R01 GM043880. The authors thank J. Allegood for skillful sphingolipid analyses and the Virginia Commonwealth University Lipidomics Core, which is supported in part by funding from NIH National Cancer Institute Cancer Center Support Grant P30CA016059 and shared resource Grant S10RR031535.

Glossary

BALbronchiolar lavage
BMbone marrow
CBCcomplete blood count
DSSdextran sodium sulfate
EAEexperimental autoimmune encephalitis
H&Ehematoxylin and eosin
LC-ESI-MS/MSliquid chromatography electrospray ionization tandem mass spectrometry
MOGmyelin oligodendrocyte glycoprotein
OVAovalbumin
S1Psphingosine-1-phosphate
SPsingle positive
SphKsphingosine kinase
Spns2Spinster 2
WTwild-type
β-Galβ-galactosidase (5-bromo-4-chloro-3-indolyl-d-galactopyranoside)

Footnotes

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

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