Paquinimod prevents development of diabetes in the non-obese diabetic (NOD) mouse

<div><p>Quinoline-3-carboxamides (Q compounds) are immunomodulatory compounds that have shown efficacy both in autoimmune disease and cancer. We have in here investigated the impact of one such compound, paquinimod, on the development of diabetes in the NOD mouse model for type I diabetes (T1D). In cohorts of NOD mice treated with paquinimod between weeks 10 to 20 of age and followed up until 40 weeks of age, we observed dose-dependent reduction in incidence of disease as well as delayed onset of disease. Further, in contrast to untreated controls, the majority of NOD mice treated from 15 weeks of age did not develop diabetes at 30 weeks of age. Importantly, these mice displayed significantly less insulitis, which correlated with selectively reduced number of splenic macrophages and splenic Ly6C^hi inflammatory monocytes at end point as compared to untreated controls. Collectively, these results demonstrate that paquinimod treatment can significantly inhibit progression of insulitis to T1D in the NOD mouse. We propose that the effect of paquinimod on disease progression may be related to the reduced number of these myeloid cell populations. Our finding also indicates that this compound could be a candidate for clinical development towards diabetes therapy in humans.</p></div

Additional file 1: Table S1. of Association of CD247 (CD3Îś) gene polymorphisms with T1D and AITD in the population of northern Sweden

Clinical characteristics of the study material. Table S2. HLA-DQB1 genotypes in the study material. Table S3. SNPs included in the GoldenGate Custom Panel. (PDF 338 kb

Reduced insulitis in paquinimod treated NOD mice.

<p>Cohorts of 15 w old mice were treated either with paquinimod (Paq; 1 mg/kg/day) or vehicle (Ctrl) as indicated. Groups of mice were sacrificed at the start of the experiment (Baseline n = 6), at 20 w of age (Ctrl n = 8; Paq n = 9) and at 30w of age (Ctrl n = 7; Paq n = 7). Serial sections of pancreatic tissue were prepared, stained with H&E and analyzed microscopically. A) Representative images are shown; scale bar in image: 100 μm. Insulitis scores B), and insulitis indexes C) are shown. In B), the extent of mononuclear cell infiltration was scored from 0 through 3. Score 0 (open bars), score 1 (light grey bars), score 2 (medium grey bars), score 3 (black bars). In C) insulitis index was calculated as described in <i>Materials and Methods</i>. Insulitis was scored by examining a minimum of 40 islets per animal. **, <i>p</i> < 0.01, ***, <i>p</i> < 0.001 by Mann Whitney U test.</p

Paquinimod treatment reduces the frequency of Ly6C^hi and F4/80⁺ cells in spleen of NOD mice.

<p>Cells from spleen and panLN of the mice in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196598#pone.0196598.g001" target="_blank">Fig 1C and 1D</a> were analyzed by flowcytometry. A), percentage of single CD19^- CD11b⁺ cells out of total viable cells, as well as percentage of Ly6C^hi inflammatory monocytes, Ly6G⁺ neutrophils, and SiglecF⁺ eosinophils among total CD11b⁺ cell population is shown both for B) spleens and C) pancreatic lymph nodes of mice. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, ****<i>p</i> < 0.0001, Mann–Whitney U test.</p

Delayed onset and reduced susceptibility to diabetes in paquinimod-treated NOD mice.

<p>Incidence of diabetes in mice treated with different doses of paquinimod (mg/kg/day; n = 10 for each dose) or vehicle (Ctrl; n = 20) from 10 to 20w of age A) or 15 to 38 w of age B). In the experiment in C) and D) NOD mice were treated with 1mg/kg/day of paquinimod or vehicle starting at 15w of age and two groups of mice (treated n = 10; controls n = 10) were sacrificed after 5 weeks of treatment (20w of age) C), and two additional groups (treated n = 10; controls n = 10) were sacrificed after 15 weeks of treatment (30w of age). Incidence of diabetes in treated groups compared to the control group (**, <i>p <</i> 0.01, ***, <i>p <</i> 0.001, by Mann Whitney U test).</p

IFN-α-secreting pDCs peak in the pancreatic islets of NOD mice at 8–9 weeks.

<p>(<b>A</b>) Infiltrating leukocytes (FVD⁻CD45⁺) from pancreatic islets were analyzed by flow cytometry for total number per mouse of T-cells (CD3⁺B220⁻CD19⁻), B-cells (CD3⁻B220⁺CD19⁺), pDCs (CD3⁻CD19⁻BST2⁺CD11c^intB220⁺), inflammatory DCs (CD3⁻CD19⁻BST2⁻CD11c^hiMHC-II⁺) and macrophages (CD3⁻CD19⁻F4/80⁺CD11b⁺MHC-II⁺) from 3 to >23 weeks of age in NOD and B6 mice (mean ± sem, <i>n</i> = 4–21 mice, 2–5 independent experiments). * p<0.05, ** p<0.005, *** p<0.0005 compared to 4-week-old B6. (<b>B</b>) Representative dot plots of FVD⁻CD45⁺CD3⁻CD19⁻ islet cells with the percentage of pDC and inflammatory DC subsets of total CD45⁺ cells indicated (mean ± sem, <i>n</i> = 4–21 mice, 2–5 independent experiments). (<b>C</b>) Expression of interferon response genes IRF7 and ISG15 is assessed by qPCR of RNA from handpicked islets of NOD (black bars) or B6 (open bars) mice at 3 and >8 weeks (<i>n</i> = 6–11 mice, 3 independent experiments). * p<0.05, ** p<0.005. (<b>D</b>) IFN-α levels in supernatants from cultured NOD islets at 4–17 weeks of age after 40h ± TLR9 ligand CpG₁₅₈₅ is assessed by ELISA (mean ± sem, <i>n</i> = 6–23 mice, 6–9 independent experiments). nd = not detected. *** p<0.0005 compared to “No” stimulation at same age.</p

Impaired recruitment of pDCs to pancreatic islets alters cytokine profile of insulitis and prevents diabetes development.

<p>(<b>A</b>) Flow cytometry analysis of leukocytes (FVD⁻CD45⁺) from handpicked pancreatic islets of fl/fl cre⁻ (black square) or fl/fl cre⁺ (open square) mice at indicated age. Cell types include pDCs (CD3⁻CD19⁻BST2⁺CD11c^intB220⁺), inflammatory DCs (CD3⁻CD19⁻BST2⁻CD11c^hiMHC-II⁺), B-cells (CD3⁻ B220⁺CD19⁺), T-cells (CD3⁺B220⁻CD19⁻). Number of cells per mouse (mean ± sem, <i>n</i> = 5–15 mice, 2–5 independent experiments). * p<0.05. (<b>B</b>) Representative dot plots of FVD⁻CD45⁺CD3⁻CD19⁻ islet cells with the percentage of pDC and inflammatory DC subsets of total CD45⁺ cells indicated (mean ± sem, <i>n</i> = 5–15 mice, 2–5 independent experiments) (<b>C</b>) IFN-α levels in supernatants from cultured fl/fl cre⁻ (black bars) or fl/fl cre⁺ (open bars) islets or cultured single cell suspensions from PaLN at 8–17 weeks of age after 40h or 24h, respectively ± TLR9 ligand CpG₁₅₈₅ is assessed by ELISA (mean ± sem, <i>n</i> = 22–24 mice, 5–6 independent experiments for islets and <i>n</i> = 13–14 mice, 4–5 independent experiments for PaLN). nd = not detected. * p<0.05. (<b>D</b>) Expression of IFN-γ is assessed by qPCR of RNA from handpicked islets of fl/fl cre⁻ (black square) and fl/fl cre⁺ (open square) mice at 3->22 weeks of age (mean ± sem, <i>n</i> = 3–9 mice, 3 independent experiments). ** p<0.005, * p<0.05 (<b>E</b>) Incidence of diabetes in fl/fl cre⁻ (black square) and fl/fl cre⁺ (open square) (<i>n</i> = 36–32). *** p<0.0001. Incidence of diabetes in +/fl cre⁺ (black circle) and +/fl cre⁻ (open circle) (<i>n</i> = 45–42). ** p = 0.0014.</p

Conditional knockout of E2-2 blocks pDC development in NOD mice.

<p>(<b>A</b>) Flow cytometry analysis of BM and spleen from 8–12 weeks old NODwt (black bars), NOD.<i>E2-2</i>^<i>fl/fl</i>-<i>CD11c</i>.<i>cre</i>⁻ (fl/fl cre⁻, striped bars) and NOD.<i>E2-2</i>^<i>fl/fl</i>-<i>CD11c</i>.<i>cre</i>⁺ (fl/fl cre⁺, open bars) mice. Data is normalized to the level of the respective cell type in NODwt. For the pDC population the total cell number in spleen is also shown. Cell subsets include pDCs (CD3⁻CD19⁻BST2⁺CD11c^intB220⁺), DCs (CD3⁻CD19⁻BST2⁻CD11c^hiMHC-II⁺), T-cells (CD3⁺B220⁻CD19⁻), B-cells (CD3⁻B220⁺CD19⁺), granulocytes (Gran) (CD3⁻CD19⁻CD11b⁺SSC^hi) and macrophages/monocytes (Mϕ/mono) (CD3⁻CD19⁻F4/80⁺CD11b⁺MHC-II⁺) (mean ± sem, <i>n</i> = 9–10 mice, 3 independent experiments). * p<0.05, ** p<0.005, *** p<0.0001. (<b>B</b>) Representative dot plots of FVD⁻CD45⁺ islet cells with percentage of pDC and inflammatory DC subsets indicated (mean ± sem, <i>n</i> = 9–10 mice, 3 independent experiments).</p

Global and 3D imaging of intact mouse CNS.

<p>(A). Spinal cords from C57BL/6 mice were stained with ASMA (red) and were further imaged by OPT. OPT scanned spinal cords were cryosectioned and imaged under a fluorescence microscope with 4× zoom (white frames). Selected regions were also imaged under a confocal microscope with 40× zoom (inlets). Nucleated cells were stained with 4,6-diamidino-2-phenylindole (DAPI). Spinal cord (B) and optic nerve (C) from B10RII mice were stained with MBP (red), and imaged by OPT. Cryosectioning was performed after OPT imaging and images were captured using a fluorescence microscope at 4× zoom. Spinal cords (D) and optic nerves (E) from C57BL/6 mice with a clinical score of 3.0 were stained with CD3 specific antibodies. OPT scanned tissues were cryosectioned and subsequently imaged under a fluorescence microscope at 4× zoom (white frames) or selected regions displaying evidence of CD3⁺ T cell infiltration were imaged using a confocal microscope at 63× zoom (inlets). CD3⁺ T cell staining is shown in red and the anatomy reconstructed from the CNS outline on the basis of the autofluorescence signal is shown in green. Scale bar: (A) Whole organ: 2 mm, 4× zoom images: 100 µm; 40× zoom image: 50 µm; (B, C) 4× zoom image: 100 µm; (D) whole organ: 2 mm, 4× zoom image: 100 µm, 40× zoom images: 50 µm, 63× zoom image: 50 µm; (E) whole organ: 2 mm, 4× zoom image: 100 µm, 63× zoom image: 50 µm.</p

Spatial assessment of neuroinflammation in B6 EAE.

<p>C57BL/6 mice were immunized with MOG_35-55/complete Freunds adjuvants (CFA)/pertussis toxin (PTX) and were observed for EAE. CNS tissue was obtained at different clinical scores and was stained for CD3⁺ T cells and imaged using OPT. The iso-surface rendered OPT images of spinal cord and optic nerve sections at different clinical scores during the progression of EAE were obtained. Infiltrating CD3⁺ T cells (red) due to the signal from the CD3 specific antibody. The reconstruction of the CNS outline is based on the autofluorescence signal (green), where the upper part of spinal cord is the cervical and thoracic region while the bottom parts are the lumbar and sacral regions. Images were generated for score 1 (n = 2), score 2 (n = 3), score 3 (n = 2). Scale bar = 2 mm.</p