Phosphorus-Based Dendrimer ABP Treats Neuroinflammation by

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Phosphorus-Based Dendrimer ABP Treats Neuroinflammation by Promoting IL-10-Producing CD4+ T Cells Myriam Hayder,†,‡,§,∥ Marjorie Varilh,†,‡,§,∥ Cédric-Olivier Turrin,⊥,# Abdelhadi Saoudi,†,‡,§,∥ Anne-Marie Caminade,⊥,# Rémy Poupot,†,‡,§,∥ and Roland S. Liblau*,†,‡,§,∥ †

Centre de Physiopathologie de Toulouse Purpan, ‡INSERM UMR1043, §CNRS UMR5282, and ∥Université Toulouse III, CHU PURPAN, BP3028 Toulouse Cedex 3, France ⊥ Laboratoire de Chimie de Coordination CNRS UPR8241-205, route de Narbonne, 31077-BP44099 Toulouse Cedex 4 France # UPS-INPT, Université de Toulouse, F31077 Toulouse Cedex 4, France S Supporting Information *

ABSTRACT: Dendrimers are polyfunctional nano-objects of perfectly defined structure that can provide innovative alternatives for the treatment of chronic inflammatory diseases, including multiple sclerosis (MS). To investigate the efficiency of a recently described amino-bis(methylene phosphonate)capped ABP dendrimer as a potential drug candidate for MS, we used the classical mouse model of MOG35−55-induced experimental autoimmune encephalomyelitis (EAE). Our study provides evidence that the ABP dendrimer prevents the development of EAE and inhibits the progression of established disease with a comparable therapeutic benefit as the approved treatment Fingolimod. We also show that the ABP dendrimer redirects the pathogenic myelin-specific CD4+ T cell response toward IL-10 production.

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INTRODUCTION Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) and the most common neuroinflammatory disorder in young adults.1,2 The disease is thought to be due to an inflammatory attack by autoreactive T cells recognizing CNS self-antigens. These autoreactive T cells migrate to the CNS and trigger further recruitment of immune cells from peripheral blood, including monocytes, thereby sustaining and amplifying an inflammatory cascade. This process leads to the destruction of the myelin sheath surrounding the axons with concomitant or subsequent axonal damage, resulting in impaired nerve conduction.3 Several therapies are available to reduce both relapse rate and disability progression in MS; however, they are associated with mild to severe adverse events, varying from transient flu-like symptoms observed with interferon-β4 to the development of progressive multifocal leukoencephalopathy with long-term natalizumab (anti-α4-integrin monoclonal antibody) treatment.5 The partial efficacy of current drugs, as well as the occurrence of such adverse events, highlights the importance of developing innovative molecules that would directly target the immunological components underlying MS pathogenesis without causing global T-cell immunosuppression. Among the recently explored strategies, nanostructures could provide an innovative therapeutic alternative for the treatment of chronic inflammatory diseases, including MS. For instance, a biodegradable nanoplatform based on poly(lactic-co-glycolic © XXXX American Chemical Society

acid) (PLG)-coupled peptide antigens has proven to be a reliable agent for the induction of antigen-specific immune tolerance.6 Dendrimers are increasingly being recognized as a valuable alternative to other poorly defined nanostructures such as polymer-based or metal-based nanoparticles. Dendrimers are polyfunctional globular nano-objects of perfectly defined structure.7 Unlike linear polymers, dendrimers are synthesized by an iterative step-by-step process that allows the creation of isomolecular species with controlled molecular size, shape, and disposition of organic moieties, leading to a perfectly defined and reproducible structure of high tunability in terms of functionalization and topology.8 To date, however, only limited studies have addressed the anti-inflammatory properties of dendrimers in animal models of acute and chronic inflammatory disorders. 9−12 In addition, these only concerned dendrimers having anti-inflammatory properties per se or pro-drug approaches with anti-inflammatory moieties grafted on, or encapsulated in, dendrimers. Poly amido-amine dendrimers ended by amine, hydroxyl, or carboxylate groups were the first dendrimers described to mitigate inflammatory responses in vivo in rat models of carrageenan-induced paw edema, granuloma, and adjuvant-induced arthritis.13 They have also been tested, as well as poly(propyleneimine) glycodenReceived: May 13, 2015 Revised: September 22, 2015

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DOI: 10.1021/acs.biomac.5b00643 Biomacromolecules XXXX, XXX, XXX−XXX

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sodium pyruvate (1 mM, Gibco), penicillin−streptomycin (1 mM, Gibco), and L-glutamine (1 mM, Gibco). Antibodies staining was performed in PBS complemented with 5% FBS. EAE Induction. C57BL/6 mice (Charles River Laboratories) and 2D2-TCR mice were housed in specific pathogen-free environment. All experimental protocols were approved by Midi-Pyrénées Ethics Committee (CEEA-122) and performed in accordance with the European Union guidelines and regulations. EAE was induced by subcutaneous immunization with 100 μg of the MOG35−55 peptide (MEVWYRSPFSRVVHLYRNGK; Mimotopes) emulsified in CFA (DIFCO Laboratories) and two intravenous injections of PTx (day 0, 200 ng; day 2, 400 ng; List Biological Laboratories). Disease scores were assessed daily on a 0 to 5 scale (0 = no abnormality; 1 = paralyzed tail; 2 = hind limb paresis; 3 = both hind limbs paralyzed; 4 = forelimbs paralyzed; and 5 = moribund). In Vivo Drug Treatment. The ABP dendrimer was injected intravenously at a dose of 10 mg/kg, whereas FTY720 was given orally at a dose of 3 mg/kg. In the prophylactic protocol, mice were treated at day 1, day 3, and then every 3 days until day 45. In the therapeutic protocol, mice were treated every 3 days or weekly from day 18 to day 45. For MOG35−55 ex vivo response experiments, C57BL/6 mice were treated at days 1, 3, and 6 and sacrificed at day 9. For IL-10 neutralization experiments, mice were treated by intraperitoneal injections of anti-IL-10R mAb (clone 1B1.3a) or IgG control (serum rat IgG) at a dose of 0.2 mg/mouse on days −1, 2, and 5. Cell Purification and Cultures. Spleens were collected from mice (nonimmunized, MOG35−55-immunized sacrificed at day 9 or 2D2TCR transgenic) and crushed in RPMI complete medium. After red blood cells lysis, CD4+ T cells were purified by negative selection using magnetic separation (Mouse Untouched CD4 Dynabeads, Invitrogen). APCs were sorted from splenocytes of nonimmunized C57BL/6 mice by depletion of T cells. In brief, splenocytes were stained with Thy1.2biotinylated antibody (clone 30-H12, BD Pharmingen) and incubated with antibiotin beads (Milteyni Biotech). Cell-beads suspension was then applied on LD column (Milteyni Biotech) and APCs were collected in the unlabeled fraction. 106 purified CD4+ T cells from either MOG35−55-immunized C57BL/6 or from 2D2-TCR transgenic mice were cocultured with 2 × 106 APCs from nonimmunized C57BL/6 mice. Cocultures as well as 106 total splenocytes from MOG35−55-immunized C57BL/6 were stimulated ex vivo during 72 h with graded concentrations of MOG35−55 (0, 0.1, 1, 10, and 100 μg/ mL). Ex Vivo Cytokine Production. Supernatants from cell cultures stimulated ex vivo during 72 h with graded concentrations of MOG35−55 were collected and IFN-γ, IL-17, and IL-10 protein concentrations were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Mouse DuoSet kits, R&D Systems). Immunohistochemistry. Brain and spinal cord from PBSperfused control mice or mice treated with ABP dendrimer every 3 days or weekly were fixed in 4% phosphate-buffered formalin and embedded in paraffin. Sections (5 μm) were stained with anti-CD3 mAb (Biolegend) to reveal the CNS-infiltrating T cells and with the pan-neuronal neurofilament marker (clone SMI-311, Biolegend) for the neuronal arborization. Antibodies and Flow Cytometry. FcR blocking with CD16/32 (24G.2) was performed on cells isolated from spleen before staining with various combinations of the following mAbs: Thy1.2-PeCy7 (532.1), F4/80-APC (BM8), CD11b-PeCy7 (M1/70), CD11c-PerCPCy5.5 (N418), class II MHC-FITC (M5/114.15.2), CD80-FITC (1610A1), and CD86-FITC (GL1) (eBiosciences); CD40-FITC (3/ 23.15.2), CD4-PB (RM4-5), CD19-APC-Cy7 (1D3), and B220-PE (RA3-6B2) (BD Pharmingen); and CD8-PerCP-Cy5.5 (YTS156.7.7) (Biolegend). Flow cytometry data were collected on a LSRII (Becton Dickinson) using the BD FACS-DIVA software and analyzed using FlowJo v.X.0.7 Software. Statistical Analyses. Data are presented as means ± SEM, and p < 0.05 was considered statistically significant. Data were analyzed using paired Student t test or Mann−Whitney U test except for EAE clinical

drimers, in neurodegenerative disorders such as models of Alzheimer’s disease and seem to inhibit the aggregation of βamyloid peptides and their detrimental activity.14 Poly(p hosph ohy drazone) (PPH) d end rimers, bearing phenoxymethyl(methyl hydrazone) branches and decorated with amines on their surface were also described to reduce the aggregation and the activity of β-amyloid peptides and the ensuing toxicity by altering the polarity of a tyrosine residue in the Aβ1−28 peptide.15,16 We previously reported that another series of PPH dendrimers decorated with phosphonate surface functions have strong immunomodulatory properties in vitro and in vivo. We have shown that amino-bis(methylene phosphonate) end groups are required at the surface of the PPH dendrimers to enable their bioactivity.17 Isosteric aminobis(methylene carboxylate) or amino-bis(methylene sulfonate) end groups are totally inactive.18 Moreover, the density of the amino-bis(methylene phosphonate) groups at the surface of the dendrimers is also a key feature.19 Finally, we recently demonstrated that the dendritic scaffold is not an inert carrier of the surface groups of the molecules because the overall 3-D conformation induced by the entire structure (including the core and the branches) is crucial for the efficiency of dendrimer nanodrugs.20 Linear analogues of polyether dendrimers have been proposed very early,21 and it has been demonstrated that the physicochemical properties (solubility, intrinsic viscosity, etc.), and of course the 3D shape are very different. In particular, among PPH dendrimers, we showed that a sixbranch anionic PPH dendrimer, capped with 12 aminobis(methylene phosphonate) end groups (ABP), dramatically inhibits inflammation and bone erosion in mouse models of experimental arthritis by inhibiting monocyte-derived osteoclasts differentiation and activity.22 Moreover, this dendrimer was also able to inhibit IL-2-driven proliferation of human CD4+ T cells.23 The synthesis process of this nano-object was previously described.24 On the basis of these findings, we reasoned that the ABP dendrimer could represent a promising drug to mitigate cellular immune responses in neuroinflammatory disorders, particularly in MS. The experimental autoimmune encephalomyelitis (EAE) is a preclinical model extensively used and is particularly useful for studying cellular and molecular mechanisms associated with CNS inflammatory tissue damage as well as for evaluating clinical utility of new potential therapies for MS.25,26 One classically used EAE model relies on immunization of C57BL/6 mice with a peptide derived from myelin oligodendrocyte glycoprotein (MOG35−55).27 Although a minor CNS myelin component, MOG has emerged as one of the most important target antigens in human inflammatory demyelinating diseases.28 Using this model, we show that ABP dendrimer could prevent the development of MOG35−55-induced EAE and inhibit the progression of established disease by promoting IL-10producing CD4+ T cells.

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EXPERIMENTAL SECTION

Material. The ABP dendrimer was synthesized as previously described24 at the ‘Laboratoire de Chimie de Coordination’ (Toulouse). FTY720 was purchased from Calbiochem (Merck distribution). The blocking anti-IL-10R monoclonal antibody was produced from clone 1B1.3a (kindly provided by Dr. Lehuen, INSERM UMR1016, Paris). Serum rat IgG was purchased from SIGMA. Both ABP dendrimer and FTY720 were prepared in Dulbecco’s phosphate-buffered saline (PBS) before administration. All culture experiments were performed at 37 °C, 5% CO2 in RPMI medium (Invitrogen) complemented with 10% FBS (Sigma-Aldrich), B


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Figure 1. ABP dendrimer inhibits the development of MOG35−55-induced EAE. (A) 7- to 9-week old C57BL/6 mice were immunized with 100 μg of MOG35−55 emulsified in CFA at day 0 and received two injections of Pertussis toxin (PTx) at days 0 and 2 (red arrows). Mice were treated i.v. with ABP dendrimer or with PBS (vehicle) at day 1, day 3, and then every 3 days over the EAE course (blue arrows). (B) Average clinical score from control (PBS-treated mice, filled squares) or ABP-treated mice (10 mg/kg, open triangles). (C) Day of disease onset for each animal in the two groups. (D) Average cumulative scores from day of disease onset to day 45 for control (PBS-treated mice, black bars) or ABP-treated mice (10 mg/ kg, white bars). Data represent the mean ± SEM of three independent experiments (n = 16/group). ***p < 0.002 as calculated with two-way ANOVA for (B) and two-tailed Mann-Whitney U test for (C) and (D). score course that was analyzed using two-way ANOVA and survival data that were analyzed using Logrank Mantel-Cox test.

induced EAE (Figure 2B,C). Moreover, none of the 32 mice treated every 3 days died and only 2 mice out of 11 treated weekly died, while 15 out of the 34 control mice died (pooled data from the experiments shown in Figure 2B,D; Supplementary Figure 1A). Improvement of the clinical scores was, however, more pronounced when mice were treated every 3 days rather than weekly. The efficiency of the two regimens of ABP treatment was also assessed by immunohistochemistry analyses performed on cerebellum and spinal cord sections at day 30 postimmunization. The anti-CD3 staining revealed a decreased number of T cells infiltrating the CNS in mice treated with ABP dendrimer, either every 3 days or weekly, in comparison with control PBS-treated mice. Moreover, the assessment of the axonal damage subsequent to EAE progression using a pan-neuronal neurofilament marker revealed a preserved neuronal arborization in the cerebellum of ABP-treated mice using the two regimens, while this neurofilament structure was damaged in control mice (Supplementary Figure 1B). Importantly, the ABP dendrimer was as efficient as FTY720 (Fingolimod) administered daily per os (Figure 2D,E). FTY720, an immunosuppressive drug acting on sphingosine-1-phosphate receptors, rapidly blocks ongoing EAE in rats33 and has recently been approved for the treatment of relapsing-remitting MS.34 We hypothesized that the ABP dendrimer could functionally modify myelin-specific CD4+ T-cell responses in the periphery. To test this hypothesis, we treated MOG35−55-immunized mice with ABP at days 1, 3, and 6 and analyzed the antigen-specific immune response at day 9. The ABP dendrimer strongly inhibited the production of IFN-γ and IL-17 by splenocytes after ex vivo stimulation with MOG35−55. This inhibition of pro-

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RESULTS AND DISCUSSION To investigate the efficiency of the ABP dendrimer as a potential drug candidate for MS, we immunized C57BL/6 mice with MOG35−55 emulsified in complete Freund’s adjuvant (CFA). This leads to CNS inflammation dominated by CD4+ T cells that are polarized to produce pro-inflammatory cytokines, in particular, IFN-γ and IL-17.29 The induction of EAE is supported by the injection of pertussis toxin (PTx) that exhibits adjuvant properties, thereby contributing to the migration of autoreactive CD4+ T cells to the CNS and progression of neurological lesions.30,31 In a first prophylactic strategy, the ABP dendrimer (or vehicle only) was used at 10 mg/kg and injected on days 1 and 3 postimmunization and then every 3 days until day 45 (Figure 1A). The ABP dendrimer potently inhibited the development of MOG35−55-induced EAE (Figure 1B−D). Indeed, not only did it delay the onset of disease but it also hampered its progression, with ABP-treated mice never exceeding a clinical score of 2. Then, we used a more clinically relevant treatment protocol to assess the efficacy of the ABP dendrimer on an established neuroinflammatory disease. To this aim, mice were treated either every 3 days or weekly with 10 mg/kg ABP (or vehicle) starting 18 days after EAE induction (Figure 2A). At this time point, the disease is established in all MOG35−55immunized mice, with an average score of 3, and autoreactive T cells have infiltrated the CNS and caused local inflammatory tissue damage.32 These experiments showed that the ABP dendrimer significantly inhibited the progression of MOG35−55C


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Figure 2. ABP dendrimer has a curative impact on established MOG35−55-induced EAE. (A) 7- to 9-week old C57BL/6 mice were immunized to develop EAE. Mice were treated i.v. with ABP dendrimer or with PBS (vehicle) from day 18 onward (blue arrows). (B) Average clinical score from control mice treated every 3 days with PBS (filled squares) or mice treated every 3 days with ABP (10 mg/kg, open triangles) or weekly ABP-treated mice (10 mg/kg, open diamonds). (C) Average cumulative scores before (days 0−18) and during (days 19−45) the treatment period for control mice (PBS-treated mice, black bars), mice treated every 3 days with ABP (10 mg/kg, white bars), or mice treated weekly with ABP (10 mg/kg, gray bars). (D) Average clinical score from control i.v. (mice injected every 3 days with PBS, filled squares), control per os (mice given daily PBS orally, filled triangles), mice treated every 3 days with ABP i.v. (10 mg/kg, open triangles) or mice treated daily with FTY720 per os (3 mg/kg, open diamonds). (E) Average cumulative scores before (days 0−18) and during (days 19−45) the treatment period from control i.v. (black bars), control per os (dark gray bars), mice treated every 3 days with ABP (10 mg/kg, white bars), or daily with FTY720 (3 mg/kg, light gray bars). Data represent the mean ± SEM of five independent experiments for (B) and (C) and of one experiment for (D) and (E) with the number of animals indicated in the graph. *p < 0.05, **p < 0.01, and ***p < 0.002 as calculated with two-way ANOVA for (B,D) and two-tailed Mann−Whitney U test for (C,E).

cultures originating from vehicle-treated mice, consistent with our results using unfractionated splenocytes (Figure 3B). Interestingly, the inhibition of IFN-γ production and increased IL-10 release were significant only when the MOG35−55immunized mice that provided CD4+ T cells, and not the mice providing the APCs, were treated by ABP dendrimer. We also observed inhibition of IL-17 production, although to a lesser extent (Figure 3C). Taken together, these results suggest that ABP treatment redirects MOG35−55-specific CD4+ T cell responses toward a regulatory phenotype characterized by IL10 production. Interestingly, the impact of the ABP dendrimer on cytokine production was more modest when only the nonimmunized mice providing APCs were treated (Supplementary Figure 2). The analysis of splenocyte numbers and cell surface markers indicated that the ABP dendrimer blunts the accumulation of APCs such as B cells, macrophages, and dendritic cells following MOG35−55-immunization (Supplementary Figure 3A) and prevents their activation (Supplementary Figure 3B). Collectively, these data suggest that the ABP

inflammatory cytokines in the periphery was associated with a sustained increase in IL-10 production (Figure 3A). Previous studies performed on experimental models of autoimmune inflammatory disorders such as EAE or arthritis showed that IL10 abrogates disease activity.35−38 Importantly, IL-10 suppresses autoreactive T cell proliferation and promotes skewing of antigen-specific T cells toward a regulatory function.37 Thus, the increased IL-10 production in our model could explain the clinical protection provided by the ABP dendrimer. IL-10 is a pleiotropic cytokine primarily produced by CD4+ T cells but also by antigen presenting cells (APCs) such as dendritic cells, B cells, and monocytes/macrophages. To determine whether IL-10 is produced by CD4+ T cells or by APCs, we stimulated purified CD4+ T cells from MOG35−55immunized mice, treated or not with ABP, with APCs from nonimmunized mice, treated or not with ABP. When both CD4+ T cells and APCs originated from mice treated with ABP, a strong decrease in IFN-γ and IL-17 release and a significant increase in IL-10 secretion were observed as compared with D


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Figure 3. ABP dendrimer redirects myelin-specific T-cell inflammatory responses toward IL-10 production. 7- to 9-week old C57BL/6 mice were immunized with 100 μg of MOG35−55 emulsified in CFA at day 0. Mice were treated i.v. with ABP dendrimer or with PBS (vehicle) at days 1, 3, and 6 and sacrificed at day 9. IFNγ, IL-17, and IL-10 release was assessed in culture supernatants of cell cultures stimulated ex vivo with graded concentrations of MOG35−55. Cell cultures consisted of (A) whole splenocytes from MOG35−55-immunized mice treated with PBS (control, filled diamonds) or with ABP (10 mg/kg, open diamonds); (B) purified CD4+ T cells from PBS-treated MOG35−55-immunized mice that were stimulated with APCs from PBS-treated nonimmunized mice (Control, filled squares) or purified CD4+ T cells from ABP-treated MOG35−55-immunized mice that were stimulated with APCs from ABP-treated nonimmunized mice (10 mg/kg, open squares); and (C) purified CD4+ T cells from MOG35−55immunized mice treated with PBS (control, filled circles) or with ABP (10 mg/kg, open circles) that were stimulated with APCs originating from PBS-treated nonimmunized mice. Data represent the mean ± SEM of four independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.002 as calculated with paired Student’s t test without correction for multiple tests.

dendrimer has a cumulative impact on both CD4+ T cells and APCs. To further investigate the underlying regulatory mechanisms triggered by ABP on APCs, we used CD4+ T cells from 2D2-Tcell receptor (TCR) transgenic mice, which express an I-Abrestricted TCR (Vα3.2-Vβ11) specific for MOG35−55.39 These CD4+ T cells were stimulated ex vivo with APCs from nonimmunized mice that were treated with the ABP dendrimer or with vehicle only. Measurements of cytokine production following ex vivo stimulation with MOG35−55 showed that APCs from mice treated with the ABP dendrimer significantly reduce the differentiation of autoreactive CD4+ T cells toward a pathogenic Th1/Th17 phenotype characterized by IFN-γ/IL17 secretion and rather promote IL-10 production (Figure 4A). These results suggest that ABP treatment targets APCs, thereby promoting the differentiation of CD4+ T cells toward regulatory IL-10-producing cells. To investigate the in vivo relevance of this potential mechanism of action of the ABP dendrimer, we used an anti-IL-10R monoclonal antibody (mAb) to neutralize IL-10 functions in vivo during EAE. It was previously reported that the blockade of IL-10 functions

leads to a more severe EAE than in mice receiving control IgG.37,40 As previously shown, ABP treatment markedly inhibited disease development; however, the anti-IL-10R antibody strongly reduced the therapeutic potential of the ABP dendrimer in MOG35−55-induced EAE (Figure 4B,C). These data further support the hypothesis that the clinical protection provided by the ABP dendrimer is, at least in part, due to its impact on IL-10-producing CD4+ T cells. The impact of ABP on peripheral autoreactive T cells could explain the protection observed in our prophylactic strategy but also could contribute to its curative effect. Indeed, by redirecting the phenotype of peripheral immune cells toward a regulatory phenotype driven by IL-10 production, the ABP dendrimer could impair the pathogenic phenotype of immune cells that cross the blood-brain barrier (BBB) or could affect the migration of these cells through the BBB. Recently, it has been proposed that nano-objects of intermediate size of a few nanometers migrate through the BBB.41−44 The curative action of ABP dendrimer could therefore be the result of a direct impact of the branched molecule on CNS cells, in addition to its impact on peripheral immune cells. Whether the ABP E


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Figure 4. ABP dendrimer promotes T-cell production of IL-10 by targeting APCs. (A) 7- to 9-week old C57BL/6 mice were treated i.v. with ABP dendrimer (10 mg/kg) or with PBS (vehicle) at days 1, 3, and 6 and sacrificed at day 9. Purified 2D2 CD4+ T cells were stimulated with APCs from PBS- (control, filled triangles) or ABP-treated nonimmunized mice (10 mg/kg, open triangles). IFNγ, IL-17, and IL-10 release was assessed in culture supernatants after ex vivo stimulation with graded concentrations of MOG35−55. Data represent the mean ± SEM of three experiments. (B) 7to 9-week old C57BL/6 mice were immunized with 100 μg of MOG35−55 emulsified in CFA and received two injections of PTx at days 0 and 2. Mice received i.p. anti-IL-10R mAb or control IgG (both at 0.2 mg/mouse) at day 1; days 2 and 5 and were treated i.v. with ABP dendrimer or with PBS (vehicle) at day 1, day 3, and then every 3 days. Average clinical scores from mice receiving control IgG and treated with PBS (filled squares), mice receiving IgG control and treated with ABP (10 mg/kg, open triangles), mice receiving anti-IL-10R mAb and treated with PBS (filled diamonds), and mice receiving anti-IL-10R mAb and treated with ABP (10 mg/kg, open diamonds) are depicted. (C) Average cumulative scores from day of disease onset to day 45 are shown for control (PBS-treated mice, black bars) or ABP-treated mice (10 mg/kg, white bars), treated or not with the blocking anti-IL10R mAb. One representative out of two independent experiments is shown. *p < 0.05; **p < 0.01 as calculated with paired Student t test without correction for multiple tests for (A), two-way ANOVA for (B), and two-tailed Mann−Whitney U test for (C).

functional polarization of autoreactive CD4+ T cells. Previous studies have documented a similar potent impact on innate immune cells of negatively charged PLG large nanoparticles, as well as their efficiency to treat EAE, to prevent disease relapses,6,48 and codelivering drugs to the inflamed tissue to generate antigen-specific response.49 Such immune-modifying functions contrast with the rather indiscriminate lymphopenia or immune cell migration blockade that underlies the mechanisms of currently approved therapies for MS such as natalizumab,50 fingolimod,51 and alemtuzumab.52 It is, however, reminiscent of the mechanism of action suggested for glatiramer acetate, a random polymer that preferentially promotes production of IL-10 and TGF-β by monocytes, thereby impacting CD4+ T cell differentiation.53 Moreover, our recent study showed that repeated injections of the ABP dendrimer did not induce immunosuppression or systemic toxicity in nonhuman primates.54 Indeed, we have followed up clinical, biochemical (ten), hematological, and immunological (more than 30), clotting, and histopathological parameters. Some of them underwent some subacute variations in the time course of the assay but were always kept in the physiological range, with a return to pretherapeutic levels within 3 days, and with no cumulative effect across the repeated injections.54 As such, the innovative approach proposed here relies on a safe nano-object with a controlled and reproducible synthesis process that targets specifically cells of the innate immune

dendrimer exerts its immuno-modulatory effect directly in the inflamed CNS tissue is currently under investigation. This possibility would open tremendous opportunities.

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CONCLUSIONS We report here two major findings. First, the ABP dendrimer is able to both prevent the development of MOG35−55-induced EAE and inhibit established disease. Second, several cellular mechanisms of action of the ABP dendrimer are operating in vivo in the mouse model of EAE: (i) the ABP dendrimer skews the cytokine production by splenocytes from immunized mice from an inflammatory pattern to an anti-inflammatory one (Figure 3A); (ii) it redirects myelin-specific CD4+ T-cell response toward IL-10 production (Figure 3B); and (iii) the latter effect is, at least in part, indirect through the action of ABP dendrimer on APCs (Figures 3C and 4). This is in agreement both with our previous observation of an enhanced IL-10 production by human CD4+ T cells in mixed leukocyte reaction upon addition of the ABP dendrimer45 and with the overall anti-inflammatory properties of the molecule toward human APCs;45,46 however, we only have preliminary insights into the molecular mechanisms of action of the ABP dendrimer on human monocytes because its specific receptor(s) is (are) not yet identified.47 The cellular mechanisms of action of the ABP dendrimer are original, as it targets and deactivates innate immune cells, thereby resulting in a profound change in the F


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Biomacromolecules

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system to provide a comparable therapeutic benefit as the approved treatment Fingolimod in a preclinical model of MS.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00643. Supplementary Figure 1. Efficiency of ABP treatment on EAE using two different regimens. It also inhibits the T cell infiltration as well as it preserves the dendritic arborisation of neurons. Supplementary Figures 2 and 3. ABP dendrimer impacts APCs. It also influences the distribution of these cells without affecting the size of the T-cell compartment and it down-regulates the activity of dendritic cells as revealed by the decreased expression of MHC class II, CD40, CD80, and CD86 markers. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Lennart T. Mars and Daniel Gonzalez-Dunia for their feedback during the study. This work was supported by Agence Nationale de la Recherche (ANR-RPIB-2011-005; grant DENDRI’MS), Association pour la Recherche sur la Sclérose en Plaques (ARSEP), Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), and the University of Toulouse.

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ABBREVIATIONS ABP, amino-bis(methylene phosphonate); APC, antigen presenting cell; BBB, blood-brain barrier; CFA, complete Freund’s adjuvant; CNS, central nervous system; mAb, monoclonal antibody; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; PBS, Dulbecco’s phosphatebuffered saline; PLG, poly(lactic-co-glycolic acid); PPH, poly(phosphohydrazone); PTx, pertussis toxin; TCR, T-cell receptor

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REFERENCES

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