Amelioration of Experimental Autoimmune Encephalomyelitis by the Quinoline-3-Carboxamide Paquinimod
2013; Elsevier BV; Volume: 182; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2013.01.032
ISSN1525-2191
AutoresSofia Helmersson, Anette Sundstedt, Adnan Deronic, Tomas Leanderson, Fredrik Ivars,
Tópico(s)Immunotherapy and Immune Responses
ResumoQuinoline-3-carboxamide compounds (Q compounds) have demonstrated efficacy in treating autoimmune disease in both humans and mice. However, the mode of action of these compounds is poorly understood. Here, we show that preventive treatment with the Q compound paquinimod (ABR-215757) during the first 5 days after induction of experimental autoimmune encephalomyelitis is sufficient to significantly ameliorate disease symptoms. Parallel cell-depletion experiments demonstrated that Ly6Chi inflammatory monocytes play an essential role in this phase. The paquinimod-induced amelioration correlated with reduced priming of antigen-specific CD4+ T cells and reduced frequency of IFN-γ– and IL-17–producing cells in draining lymph nodes. Importantly, the treatment did not inhibit T-cell division per se. In mice with established experimental autoimmune encephalomyelitis, the numbers of Ly6Chi CD115+ inflammatory monocytes and CD11b+CD11c+ dendritic cells (DCs) were reduced in spleen, but not in bone marrow or draining lymph nodes of treated mice. Inflammatory monocyte–derived DCs and CD4+ T cells were also reduced in the brain. In contrast, there was no decrease in DC subsets previously shown to be critical for effector CD4+ T-cell development in lymph nodes. Taken together, these data indicate that preventive treatment with paquinimod ameliorates experimental autoimmune encephalomyelitis by reducing effector T-cell priming and, on prolonged treatment, displays a selective effect by decreasing distinct subpopulations of splenic CD11b+ myeloid cells. Quinoline-3-carboxamide compounds (Q compounds) have demonstrated efficacy in treating autoimmune disease in both humans and mice. However, the mode of action of these compounds is poorly understood. Here, we show that preventive treatment with the Q compound paquinimod (ABR-215757) during the first 5 days after induction of experimental autoimmune encephalomyelitis is sufficient to significantly ameliorate disease symptoms. Parallel cell-depletion experiments demonstrated that Ly6Chi inflammatory monocytes play an essential role in this phase. The paquinimod-induced amelioration correlated with reduced priming of antigen-specific CD4+ T cells and reduced frequency of IFN-γ– and IL-17–producing cells in draining lymph nodes. Importantly, the treatment did not inhibit T-cell division per se. In mice with established experimental autoimmune encephalomyelitis, the numbers of Ly6Chi CD115+ inflammatory monocytes and CD11b+CD11c+ dendritic cells (DCs) were reduced in spleen, but not in bone marrow or draining lymph nodes of treated mice. Inflammatory monocyte–derived DCs and CD4+ T cells were also reduced in the brain. In contrast, there was no decrease in DC subsets previously shown to be critical for effector CD4+ T-cell development in lymph nodes. Taken together, these data indicate that preventive treatment with paquinimod ameliorates experimental autoimmune encephalomyelitis by reducing effector T-cell priming and, on prolonged treatment, displays a selective effect by decreasing distinct subpopulations of splenic CD11b+ myeloid cells. Quinoline-3-carboxamides (Q compounds) are currently in clinical development for the treatment of multiple sclerosis,1Comi G. Abramsky O. Arbizu T. Boyko A. Gold R. Havrdová E. Komoly S. Selmaj K. Sharrack B. Filippi M. LAQ/5063 Study GroupOral laquinimod in patients with relapsing-remitting multiple sclerosis: 36-week double-blind active extension of the multi-centre, randomized, double-blind, parallel-group placebo-controlled study.Mult Scler. 2010; 16: 1360-1366Crossref PubMed Scopus (53) Google Scholar, 2Comi G. Pulizzi A. Rovaris M. Abramsky O. Arbizu T. 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In the present study, we used the myelin oligodendrocyte glycoprotein (MOG)–induced EAE model to study the possible effects of paquinimod treatment on cells involved in the induction phase of the disease, in particular CD4+ T cells, various antigen-presenting cells (APCs), and monocytes. C57BL/6 (B6) mice were purchased from Taconic Farms (Ejby, Denmark). (TCR)-Vβ3 transgenic mice were originally obtained from Mark M. Davis (Stanford University), and ovalbumin-specific TCR (OT-II) transgenic mice were bred at the BMC animal facility at Lund University. The mice were treated with the Q compound paquinimod (a gift from Active Biotech AB, Lund, Sweden) dissolved in drinking water available ad libitum. Except as otherwise stated, the compound was used at a dosage of 25 mg/kg per day, starting on the day before immunization. The experiments involving use of animals were performed in accordance with approved local ethical guidelines. Mice were immunized subcutaneously at the base of the tail with 50 μg MOG35-55 peptide (Schafer-N, Copenhagen, Denmark) in PBS emulsified in CFA. In some experiments, mice were immunized with PBS/CFA emulsion without added protein antigen. To induce development of EAE, the MOG-peptide–immunized B6 mice were also injected twice (on the day of immunization and 2 days later) with 200 ng pertussis toxin (List Biological Laboratories, Campbell, CA) in PBS. Mice were injected intravenously with 500 μg of either anti–Gr-1 antibody (RB6-8C5), anti-Ly6G antibody (1A8), or isotype control (MPC-11) (all from Bio X Cell, West Lebanon, NH) on the day before EAE induction. Mice were scored daily for development of EAE, starting at day 8 or 9 after immunization. Neurological defects were scored as 0 (no disease), 1 (weakness of the tail), 2 (flaccid tail), 3 (hind limb weakness), 4 (paralysis in one hind limb), 5 (total paralysis of hind limbs), 6 (forelimb weakness), 7 (forelimb paralysis), or 8 (dead). Lymph nodes or spleens were dissected, and single-cell suspensions were prepared. For isolation of splenic or lymph node DCs, the organs were cut into small pieces and treated with collagenase IV/DNase before preparation of single-cells suspensions. The single-cell suspensions were passed through 70-μm cell strainers before and after a wash. DCs and CD4+ T cells were enriched using magnetic columns and CD11c or CD4-conjugated paramagnetic beads, respectively, according to the manufacturer's protocols (Miltenyi Biotec, Bergisch Gladbach, Germany; Auburn, CA). In some experiments, lymph node cells were depleted of CD19+ and CD4+ cells by using CD4- and CD19-conjugated paramagnetic beads (Miltenyi Biotec) before analysis. To prepare cells from the CNS, brain and spinal cord were dissected after intracardial perfusion with PBS. The brain and spinal cord were squeezed through a stainless steel mesh and then treated with collagenase/DNase for 40 minutes at 37°C. The single-cell suspension was passed through a 100-μm cell strainer before washing. Leukocytes were isolated by centrifugation (400 × g for 20 minutes) on a 30% to 70% Percoll (GE Healthcare Bio-Sciences, Uppsala, Sweden) gradient. Leukocytes were collected from the 30% to 70% interphase and were washed once before further analysis by flow cytometry. Cells were stained with antibodies using standard protocols. The following antibodies were used: CD11b-Alexa700 (clone M1/70), CD11c-APC/Cy7 (clone N418), CD11c-phycoerythrin (PE)/Cy7 (clone N418), Ly6G-fluorescein isothiocyanate (FITC) (clone 1A8), B220-PerCP-Cy5.5 (clone RA3-6B2), CD45.2-APC/Cy7 (clone 104), CD4-PacificBlue (clone RM4-5), MHCII-PacificBlue (clone M5/114.15.2), CD62L-PE/Cy7 (clone MEL-14), F4/80-APC/Cy7 (clone BM8), CD40-PE (clone 1C10), CD64-PE (clone X54-5171.1), and CD3-PerCP-Cy5.5 (clone 145-2C11) from BioLegend (San Diego, CA); Ly6C-biotin (clone AL-21), SiglecF-PE (clone E50-2440), TCRβ-APC (clone H57-597), CD115-APC (clone AFS98), CD80-FITC (clone 1610-A1), and CD86-APC (clone GL1) from eBioscience (San Diego, CA); and streptavidin-BD HorizonV500, CD44-PE (clone IM7), CD103-bio (clone M290), CD103-PE (clone M290), and Ly6C-bio (clone AL-21) from BD Biosciences (San Jose, CA). CD8α-FITC (clone 53.6.72) was prepared in-house. Dead cells were excluded from analysis by staining the cells with either propidium iodide or fixable red fluorescent reactive dye (Life Technologies–Invitrogen, Carlsbad, CA). To study in vivo T-cell proliferation, immunized mice were injected with 2 mg/day 5-bromo-2′-deoxyuridine (BrdU; Sigma-Aldrich, St. Louis, MO) i.p. for 2 to 4 days. BrdU incorporation was studied using a FITC BrdU flow kit (BD Biosciences). Alternatively, to study in vivo T-cell proliferation, splenocytes were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), using standard protocols. The number of divisions was expressed as the proliferation index, which was determined using ModFit LT software version 2.0 (Verity Software House, Topsham, ME). Cells were analyzed using an LSR II flow cytometer (BD Biosciences) with FlowJo software version 9.0 (Tree Star, Ashland, OR). Inguinal draining lymph nodes (dLNs) and spleen were dissected, and single-cell suspensions were prepared. Total cells were cultured with 50 ng/mL phorbol 12-myristate 13-acetate, 500 ng/mL ionomycin, and 10 μg/mL brefeldin A (Sigma-Aldrich) for 4 hours. Cells were then washed and stained with antibodies for intracellular cytokines using fixation/permeabilization diluent, fixation/permeabilization buffer, and permeabilization buffer (eBioscience) according to stand
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