4-methylumbelliferone (4-MU) is an oral drug that inhibits synthesis of hyaluronan, an extracellular matrix polymer implicated in autoimmunity. In the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), 4-MU drives the polarization of T cells away from a Th1 phenotype, associated with disease progression, and toward a FoxP3+ regulatory T-cell phenotype, associated with disease prevention. Moreover, 4-MU inhibits the reactive response of astrocytes, immunocompetent resident cells of the CNS, and prevents trafficking of activated T cells to the CNS. These data suggest that the extracellular matrix, and hyaluronan in particular, may be an active contributor to autoimmune pathogenesis in EAE and, furthermore, that 4-MU is a promising strategy for treatment of CNS autoimmunity.
The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3+ regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS.
Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). In MS and its mouse model, experimental autoimmune encephalomyelitis (EAE), lymphocyte infiltration is associated with destruction of myelin, often leading to profound neurologic impairments (1, 2). Although much is known about the cellular factors that contribute to disease progression in CNS autoimmunity, the contributions of the extracellular matrix (ECM) remain relatively poorly understood.
One ECM component that is abundant at sites of autoimmune inflammation is hyaluronan (HA), a glycosaminoglycan that contributes to tissue structure as well as cellular migration, signaling, and development (3). HA is synthesized by a class of integral membrane proteins called HA synthases (HAS1–3) and extruded through the cell membrane into the extracellular space (4). HA is highly abundant within chronically inflamed tissues, including wounds, liver cirrhosis, type 2 diabetes, and atherosclerotic plaques (3). Typically, HA at these sites has proinflammatory effects, driving dendritic cell maturation and promoting phagocytosis, antigen presentation, and T-cell activation (3⇓⇓⇓⇓–8). HA is also known to accumulate at sites of autoimmune inflammation, including pancreatic islets in type 1 diabetes (9), joints in rheumatoid arthritis (10), and other autoimmune diseases. In particular, HA fragments are known to promote inflammatory responses via interactions with Toll-like receptors (TLRs), including TLR2 and TLR4 (11, 12), and to function as a damage-associated molecular pattern molecule (13).
In MS, HA deposits are present in areas of demyelination (14). In the healthy CNS, astrocytes produce low levels of HA, depositing it as ECM complexes in the spaces between myelinated axons and between myelin sheaths and astrocyte processes (15). Upon injury, however, reactive astrocytes are the main producers of abundant amounts of HA, which accumulate in the damaged areas (14, 16, 17). As such, HA is highly abundant within demyelinated lesions in MS and in EAE, where it has been implicated in the extravasation of activated T cells into the CNS (14, 18).
Given these associations with inflammation and autoimmunity, the suppression of HA production has been explored as a therapeutic strategy. The coumarin derivative 4-methylumbelliferone (4-MU, Hymecromone) in particular has been shown to inhibit HA production in vitro and in vivo (reviewed in ref. 19). 4-MU functions as a competitive substrate for UDP-glucuronyltransferase, an enzyme involved in HA synthesis (Fig. S1) (20, 21). In addition, it has been shown that 4-MU treatment lowers HAS expression, thus reducing the production of HA (21). A recent report has demonstrated that 4-MU decreases the incidence of autoimmunity in EAE (22). However, the impact of 4-MU on astrogliosis, and lymphocyte polarization and trafficking has not been fully delineated.
Postulated 4-MU mechanism of HA synthesis inhibition. HA is produced by HAS from the precursors UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetyl-glucosamine (UDP-GlcNAc). These are generated by the transfer of a UDP-residue to N-acetylglucosamine and glucuronic acid, respectively, via UDP-glucuronyltransferase. The availability of UDP-GlcUA and UDP-GlcNAc thereby controls HA synthesis. By enzymatic conjugation to GlcUA, 4-MU depletes cytosolic levels of UDP-GlcUA, thus inhibiting the synthesis of HA, dependent on this precursor. In addition to this mechanism, it has been shown that 4-MU treatment lowers HAS expression.
Here, we have treated EAE mice with 4-MU and assessed the effect of this treatment on the development and progression of EAE. In particular, we have tested the hypothesis that 4-MU treatment prevents the development of a pathogenic T-cell response and inhibits the trafficking of autoreactive T cells through lymphoid organs and into CNS tissue.
4-MU Treatment Prevents and Ameliorates EAE.
We first assessed the impact of oral 4-MU treatment on clinical symptoms of myelin oligodendrocyte glycoprotein (35–55) (MOG35–55)–induced EAE in C57BL/6 mice. Treatment with 4-MU, started before induction of disease (pretreatment protocol) or after induction of the disease but before the onset of symptoms (treatment protocol), significantly reduced the incidence, delayed the onset, and decreased the severity of EAE (Fig. 1 A and B and Table S1). The incidence of EAE was 28% and 40% after pretreatment and treatment, respectively, compared with 90% in untreated animals. In addition, disease onset was delayed by 8.9 and 11.1 d. Severity was reduced to an average score of 0.03 ± 0.03 (peak score 1.0 ± 0.0) and 0.15 ± 0.11 (peak score 2.0 ± 0.6), after 4-MU pretreatment or treatment, compared with an average disease severity of 2.2 ± 0.2 (peak score 3.7 ± 0.2) in untreated animals. Moreover, an intervention protocol where 4-MU treatment was started after onset of symptoms (when animals reached a score of 1) significantly reduced disease severity to an average score of 1.4 ± 0.2 (peak score 2.6 ± 0.2) (Fig. 1C and Table S1), indicating that 4-MU ameliorates established disease.
Oral 4-MU treatment prevents and reverses autoimmune demyelination. (A–C) EAE scores of mice treated with 4-MU (5% wt/wt in the chow) started before induction of disease (A, pretreatment protocol), after induction of disease, before onset of symptoms (B, treatment protocol), or after onset of disease (when mice reached a score of 1; C, intervention protocol) induced by immunization with MOG35–55. (D) EAE scores of mice treated with 4-MU started 14 d before immunization continuously or until day 21 after immunization. Shown are mean scores ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, Mann–Whitney comparing treated mice with untreated mice (n = 10). Arrows indicate timing of disease induction (black arrows depicting immunization at day 0 and gray arrows the second PTX injection at day 2), and gray shaded boxes indicate the duration of 4-MU treatment.
To determine whether ongoing 4-MU treatment is required for extended disease prevention, we stopped treatment at day 21 after induction of disease. At this time point, treated animals showed no signs of disease, whereas untreated animals were at peak of disease. Cessation of treatment led to an immediate manifestation of disease, displaying the same kinetics as untreated disease at first onset (Fig. 1D). This indicates that the therapeutic effect of 4-MU requires continuous treatment.
Summary of treatment effect of 4-MU in EAE (mean values ± SEM)
4-MU Treatment Skews Th Cell Profiles Away from Th1.
To determine how 4-MU treatment affected the inflammatory process in EAE, we analyzed the profile of infiltrating inflammatory cells in the spinal cord of 4-MU–treated and untreated animals using flow cytometry. Correlating with disease severity, total numbers of infiltrating inflammatory cells at peak of disease (day 22 after immunization) were significantly reduced by all 4-MU treatment protocols (Fig. 2A and Fig. S2). This reduction was mostly due to a reduction in the number of infiltrating CD4+ T cells (Fig. 2B and Fig. S3A). We therefore further assessed infiltrating Th1, Th17, and Th2 subsets in the spinal cord, using intracellular staining for IFN-g, IL-17, and IL-4, respectively. 4-MU treatment mostly reduced the number of infiltrating Th1 cells, whereas it only mildly reduced Th17 cell numbers and did not affect Th2 cell numbers (Fig. 2C and Fig. S3B).
4-MU treatment alters Th cell profiles. (A) Total number of cells, (B) total number of CD4+ cells, and (C) number of IFN-g–, IL-17–, and IL-4–producing cells detected in spinal cord tissue of naïve mice and EAE mice left untreated or after treatment with 4-MU using the treatment protocols described in Fig. 1. Cells were isolated from pooled spinal cords of five animals reflecting the average score per treatment group at peak of disease (day 22 after induction of disease) and analyzed by flow cytometry after intracellular cytokine staining. (D) Proliferation of splenocytes isolated from naïve mice and EAE mice left untreated, or after the various 4-MU treatment protocols at peak of disease, as measured by thymidine incorporation. Shown are individual values and mean ± SEM; *P < 0.05, unpaired t test, n = 4–5. (E) Number of IFNg–, IL-17–, and IL-4–producing cells in spleens at peak of disease (day 20) and (F) early in disease (day 3 after induction of disease). Splenocytes were isolated from five separate animals reflecting the average score per treatment group and analyzed by flow cytometry after intracellular cytokine staining. Shown are individual values and mean ± SEM; *P < 0.05, Mann–Whitney.
Clinical scores of animals used in flow cytometry analysis of T-cell populations in the spinal cord, spleen, and lymph nodes. Shown are disease scores of individual animals and the mean ± SEM at the time of harvest (peak of disease, day 22 after immunization).
Breakdown of cell populations infiltrated into the spinal cord. (A) Overview of CD4+, CD8+, and macrophage cells detected in spinal cord tissue of naïve mice and EAE mice left untreated or after treatment with 4-MU using the treatment protocols described in Fig. 1. (B) Overview of the number of IFN-g–, IL-17–, and IL-4–producing cells within the CD4+ cell population depicted in A. Cells were isolated from pooled spinal cords of five animals reflecting the average score per treatment group at peak of disease (day 22 after induction of disease) and analyzed by flow cytometry after cell surface and intracellular cytokine staining.
We further examined the effects of 4-MU on immunity within secondary lymphoid organs. Splenocyte proliferation after in vitro restimulation was reduced by all treatment protocols at peak of disease (Fig. 2D). We did not observe skewing of the Th response away from Th1 in secondary lymphoid organs at peak of disease (Fig. 2E). However, it did manifest at these sites early in the disease, before inflammatory cells had migrated to the CNS (on day 3 after immunization; Fig. 2F).
Consistent with these effects on peripheral Th subset polarization in the EAE model, we observed similar skewing away from IFN-g, IL-17, and IL-6 production and toward production of IL-4 and IL-5 in nonimmunized mice treated with 4-MU. In addition, treatment of these animals induced an increase in cells expressing GATA3, the master transcription factor of Th2 polarization (Fig. S4).
4-MU treatment alters Th cell polarization toward an anti-inflammatory profile in naïve mice. (A) Production of IFN-g, IL-17, IL-6, IL-10, IL-4, and IL-5, measured by ELISA, by splenocytes from naïve C57BL/6 or BALB/c mice either untreated or after 4-MU treatment (a minimum of 2 wk), stimulated in vitro with Con A (2 μg/mL). *P < 0.05, unpaired t test, n = 5. (B) Number of Th1, Th2, and Th17 cells, as assessed by expression of Tbet, RORgT, and GATA3, respectively, in the spleen of naïve C57BL/6 mice either untreated or after 4-MU treatment (a minimum of 2 wk). Splenocytes were isolated from five separate animals per treatment group and analyzed by flow cytometry after intracellular staining for transcription factors. Shown are individual values and mean ± SEM; *P < 0.05, Mann–Whitney.
Together, these data indicate that 4-MU limits Th1 polarization in both CNS autoimmunity as well as in peripheral, noninflamed tissues. However, the pace of these effects differs depending on the tissue in question.
4-MU Treatment Alters Treg Profiles.
Given the reduced inflammation and improvements in disease outcomes, we asked whether 4-MU treatment enhanced immune regulation. We indeed observed that 4-MU treatment increased the infiltration of Treg into the spinal cord at peak of disease (Fig. 3A). Treg numbers returned to untreated levels when treatment was withdrawn (Fig. 3B), showing that continued inhibition of HA synthesis is necessary for the effect of 4-MU on Treg.
4-MU treatment supports the activation and persistence of Treg and their infiltration into the spinal cord. (A) Numbers of Foxp3+ Treg in spinal cord tissue at peak of disease (day 22) after the various 4-MU treatment protocols and (B) at day 34, 14 d after cessation of treatment (at day 21 postimmunization, 4-MU > control) compared with continuous treatment until day 34 (4-MU). (C) Percentage of Foxp3+ Treg in the spleen and lymph nodes (LNs) at peak of disease. (D) Percentage and expression levels of GITR (geometric MFI) of CD25high/GITR-expressing CD4+ cells in the spleen and LNs at peak of disease. Shown are individual values and mean ± SEM; *P < 0.05, Mann–Whitney. (E) Percentage of Foxp3+ cells and expression of Foxp3 and GITR in in vitro-induced Treg. 4-MU treatment was started 24 h after induction of Treg from CD4+ T cells using CD3/CD28 stimulation in the presence of IL-2 (100 IU/mL) and TGF-β (50 ng/mL), and cells were analyzed 48 h later. Shown are mean values + SEM; *P < 0.05, **P < 0.01, Mann–Whitney.
Concomitant with higher numbers of Treg in the spinal cord, we observed higher numbers of Foxp3+ Treg in lymphoid tissue in 4-MU–treated animals at peak of disease and in naïve animals treated with 4-MU (Fig. 3C and Fig. S5 A and B). In addition, lymphoid tissue contained a higher proportion of CD4+CD25+ T cells that also expressed the Treg costimulatory receptor glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), a regulator of Treg function (23, 24), both at peak of disease and in naïve-treated animals (Fig. 3D and Fig. S5B).
4-MU treatment increases Treg numbers in lymphoid tissue in EAE and in naïve animals. (A) Numbers of CD25+ CD4+ Treg in spinal cord tissue at peak of disease (day 22) after the various 4-MU treatment protocols. (B) Percentage of Foxp3+ Treg and percentage and expression levels of GITR (geometric MFI) of CD25high/GITR-expressing CD4+ cells in the spleen and lymph nodes (LNs) of naïve C57BL/6 mice either untreated or after 4-MU treatment (a minimum of 2 wk). Shown are individual values and mean ± SEM; *P < 0.05, Mann–Whitney.
To further explore the effect of 4-MU on the phenotype of Treg, we assessed the impact of 4-MU on Treg induction in vitro. 4-MU treatment initiated 24 h after stimulation of GFP–Foxp3-negative CD4+ T cells with anti-CD3/CD28 in the presence of IL2 and TGF-β increased the fraction of induced Treg and their expression of Foxp3 as well as GITR (Fig. 3E).
Together these data support the conclusion that 4-MU treatment promotes the differentiation of Foxp3+ Treg.
4-MU Treatment Impairs T-Cell Trafficking.
Given the observation that T-cell infiltration into the spinal cord was reduced, we investigated how 4-MU treatment impacts trafficking of T cells. To test this, we performed adoptive transfer of autoreactive, MOG-specific CD4+ T cells harvested from immunized, luciferase-expressing donor mice into albino C57BL/6 mice.
Trafficking of cells to the spleen occurred early in untreated mice and was visible in all mice 3 d after transfer, whereas this was delayed and more transient in 4-MU–treated mice. In these animals, cells were only visible in the spleen in three out of four mice and mostly after 7 d (Fig. 4 A and B, arrows, and Fig. S6). Moreover, cells did not persist in the spleens of 4-MU–treated mice as long as in untreated mice. Cells were visible in the spleen at three time points (with 3–4-d intervals) in untreated mice, whereas they were visible at two time points in 4-MU–treated animals (Fig. 4 A and B, arrows, and Fig. S6). Trafficking to lymph nodes was likewise observed in some of the untreated mice after 3–7 d but not in the 4-MU–treated mice (Fig. 4 A and B, arrow heads, and Fig. S6). Signal of transferred cells was lost by day 14 and reappeared in lymph nodes of some untreated mice by day 19, persisting until day 42, but did not reappear in 4-MU–treated mice (Fig. S6).
The glycosaminoglycan hyaluronan is important in many tissuerepair processes. We have investigated the synthesis of hyaluronan in a panel of cell lines of fibroblastic and epithelial origin in response to PDGF (platelet-derived growth factor)-BB and other growth factors. Human dermal fibroblasts exhibited the highest hyaluronan-synthesizing activity in response to PDGF-BB. Analysis of HAS (hyaluronan synthase) and HYAL (hyaluronidase) mRNA expression showed that PDGF-BB treatment induced a 3-fold increase in the already high level of HAS2 mRNA, and increases in HAS1 and HYAL1 mRNA, whereas the levels of HAS3 and HYAL2 mRNA were not affected. Furthermore, PDGF-BB also increased the amount and activity of HAS2 protein, but not of HYAL1 and HYAL2 proteins. Using inhibitors for MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1/2] (U0126) and for PI3K (phosphoinositide 3-kinase) (LY294002), as well as the SN50 inhibitor, which prevents translocation of the active NF-κB (nuclear factor κB) to the nucleus, we observed a complete inhibition of both HAS2 transcriptional activity and hyaluronan synthesis, whereas inhibitors of other signalling pathways were without any significant effect. TGF-β1 (transforming growth factor-β1) did not increase the activity of hyaluronan synthesis in dermal fibroblasts, but increased the activity of HYALs. Importantly, inhibition of hyaluronan binding to its receptor CD44 by the monoclonal antibody Hermes-1, inhibited PDGF-BB-stimulated [3H]thymidine incorporation of dermal fibroblasts. We conclude that the ERK MAPK and PI3K signalling pathways are necessary for the regulation of hyaluronan synthesis by PDGF-BB, and that prevention of its binding to CD44 inhibits PDGF-BB-induced cell growth.
Abbreviations: BCA, bicinchoninic acid; bFGF, basic fibroblast growth factor; CREB, cAMP-response-element-binding protein; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; ERK, extracellular-signal-regulated kinase; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HABP, hyaluronan-binding protein; b-HABP, biotinylated HABP; HAS, hyaluronan synthase; HEK-293, human embryonic kidney; HRP, horseradish peroxidase; HYAL, hyaluronidase; IκB, inhibitor of nuclear factor κB; MAPK, mitogen-activated protein kinase; MEF, mouse embryonic fibroblast; MEK, MAPK/ERK kinase; NF-κB, nuclear factor κB; PDGF, platelet-derived growth factor; PI3K, phosphoinositide 3-kinase; STAT, signal transducer and activatory of transcription; TBS-T, Tris-buffered saline with Tween 20; TGF-β1, transforming growth factor-β1
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