Association between BPF and OCT parameters in patients with RRMS.

<p>Patients (individual eyes) are labeled according to the history of optic neuritis (ON). Lines are derived from linear regression analyses with R² given in parentheses. Statistical significance level was calculated by Generalized Estimating Equation models controlling for the history of ON. A) Retinal nerve fiber layer thickness (RNFLT) vs. BPF (0.073, p = 0.019). B) Total macular volume (TMV) vs. BPF (0.113, p = 0.001).</p

Description of study cohort with demographic and disease parameters.

<p>RRMS = relapsing remitting Multiple sclerosis; (N)ON = (non) optic neuritis; SD = standard deviation, BPF = brain parenchymal fraction, RNFLT = retinal nerve fiber layer thickness, TMV = total macular volume.</p

Generalized Estimating Equations for the association of RNFLT with BPF as primary endpoint.

<p>Results from GEEs with RNFLT and age as independent variables and controlling for history of optic neuritis and BPF as dependent variable. The standardized Beta was calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018132#s2" target="_blank">methods</a> section. RNFLT = retinal nerve fiber layer thickness, ON = history of optic neuritis, CI = confidence interval.</p

Modulation of Dendritic Cell Immunobiology via Inhibition of 3-Hydroxy-3-Methylglutaryl-CoA (HMG-CoA) Reductase

<div><p>The maturation status of dendritic cells determines whether interacting T cells are activated or if they become tolerant. Previously we could induce T cell tolerance by applying a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor (HMGCRI) atorvastatin, which also modulates MHC class II expression and has therapeutic potential in autoimmune disease. Here, we aimed at elucidating the impact of this therapeutic strategy on T cell differentiation as a consequence of alterations in dendritic cell function. We investigated the effect of HMGCRI during differentiation of peripheral human monocytes and murine bone marrow precursors to immature DC <i>in vitro</i> and assessed their phenotype. To examine the stimulatory and tolerogenic capacity of these modulated immature dendritic cells, we measured proliferation and suppressive function of CD4+ T cells after stimulation with the modulated immature dendritic cells. We found that an HMGCRI, atorvastatin, prevents dendrite formation during the generation of immature dendritic cells. The modulated immature dendritic cells had a diminished capacity to take up and present antigen as well as to induce an immune response. Of note, the consequence was an increased capacity to differentiate naïve T cells towards a suppressor phenotype that is less sensitive to proinflammatory stimuli and can effectively inhibit the proliferation of T effector cells <i>in vitro</i>. Thus, manipulation of antigen-presenting cells by HMGCRI contributes to an attenuated immune response as shown by promotion of T cells with suppressive capacities.</p></div

Expression of MHCII, costimulatory and maturation markers is attenuated in aiDCs.

<p>(A) Expression of surface markers CD1c, CD11b, CD11c, CD14, CD40, CD80 and CD86 on human iDC cultured in the presence of atorvastatin was measured by flow cytometry after the differentiation period (day 7) and compared with surface marker expression of untreated iDC and undifferentiated monocytes from peripheral blood together with isotype controls for untreated iDC and monocytes. The data show relative expression of surface molecules compared to untreated iDC derived from three independent experiments (mean ± SD). (B) Expression of surface markers CD11b, CD11c, CD40, CD80, CD86 and MHC class II of murine iDC cultured in the presence of atorvastatin was measured by flow cytometry after the differentiation period (day 10) and compared with surface marker expression of untreated iDC and isotype controls for untreated iDC. The data show relative expression of surface molecules compared to untreated iDC derived from three independent experiments (mean ± SD).</p

Influence of atorvastatin treatment during DC differentiation on f-actin expression.

<p>Human monocytes and murine BMC were differentiated into iDC in the presence of different atorvastatin concentrations: human iDC (A) untreated, 0.5 µM, 1 µM, 2 µM, 5 µM and 5 µM atorvastatin in the presence of 200 µM mevalonate. Murine iDC (B) untreated, 0.5 µM, 1 µM, 2 µM and 5 µM atorvastatin in the presence of 200 µM mevalonate. DC were stained for f-actin with rhodamin-coupled phalloidin (red) and nuclei were stained with Hoechst 33342 (blue). Human iDC were additionally stained for CD11c (green). The insert panels highlight representative structural cell features at an additional threefold magnification. (C) The level of actin polymerization in murine iDC was quantified by flow cytometry. The fluorescence intensity of phalloidin-FITC bound to f-actin was analyzed for different atorvastatin concentrations and mevalonate. The data show relative f-actin expression compared to untreated iDC derived from five independent experiments (mean + SD).</p

iDC generated in the presence of atorvastatin have a decreased capacity to take up antigen and to initiate an allogenic response.

<p>(A) FITC-dextran uptake was measured in human iDC by flow cytometry. Data show the mean relative fluorescence values for different atorvastatin concentrations compared to untreated iDC (mean + SD) for FITC-dextran uptake at 37°C (left columns) and 0°C (right dashed columns). (B) Human iDC were generated from monocytes in the presence of different atorvastatin concentrations and subjected to MLR with allogeneic T cells in different iDC-to-T cell ratios (iDC alone, 1:10, 1:20, 1:40, 1:80). MLR strength was measured by ³H-thymidine incorporation of the co-culture. Plotted is the mean ³H-thymidine incorporation in counts per minute (cpm), these data are representative of three independent experiments. Data are representative of three independent experiments. (C) Mouse iDC (C57Bl/6) were generated from BMC in the presence of different atorvastatin concentrations and subjected to MLR with allogeneic spleen cells (SJL/N) in different iDC to spleen cell ratios (iDC alone, 1:10, 1:20, 1:40). MLR strength was measured by ³H-thymidine incorporation of the co-culture. Plotted is the mean ³H-thymidine incorporation in counts per minute (cpm), these data are representative of three independent experiments. (D) Ova-loaded iDC and aiDC were injected subcutaneously. Ova-specific OT-II T cell response of draining lymph node cells was measured by ³H-thymidine incorporation. Plotted are the mean stimulation indices as ratio of ³H-thymidine incorporation compared to unstimulated lymph node cells alone. Data are representative of three independent experiments.</p

Atorvastatin primes iDC to produce IL-10 and to generate T cells with a greater suppressive capacity.

<p>(A) Intracellular IL-10 expression in C57Bl/6-derived iDC and aiDC, unstimulated and stimulated with PMA-ionomycin, was measured by flow cytometry. Quadrants were set according to unstained controls. (B) Atorvastatin treated and untreated iDC were used to generate a regulatory T cell population by repetitive stimulation of allogeneic naïve CD4 T cells in the presence of regulatory cytokines. The regulatory capacity was assessed by the suppression of proliferation of pre-activated CD4 effector cells. Shown are the changes of effector T cell proliferation (in percent) for different suppressor T cell to T effector cell (targets) ratios and different atorvastatin concentrations (untreated, 2 µM and 10 µM) used during generation of iDC. Representative experiments of at least three are shown. p<0.05 was considered statistically significant, asterisks represent ** = p<0.01, *** = p<0.001.</p

Correlation of RNFLT with BPF and ¹H-MRS parameters.

<p>a) Depicted is the average RNFLT, every symbol representing a single eye examined together with the corresponding BPF values. The symbols represent the patient's previous history of optic neuritis (open circles – no previous optic neuritis, grey squares - unilateral optic neuritis, black triangles – bilateral optic neuritis) A linear correlation function was calculated by a Generalised Linear Model to account for intra-individual inter-eye relationships (p = 0.001). b) Mean BPF was calculated for three groups that were defined based on their previous history of optic neuritis (white bar– no previous optic neuritis, grey bar - unilateral optic neuritis, black bar – bilateral optic neuritis). The (-) symbol indicates a trend, but a missing significant correlation of group differences as calculated by ANOVA (p = 0.055). Error bars represent 2× standard error of the mean (SEM). c) RNFLT averages are shown in relation to corresponding NAA concentrations in the visual cortex (VC). The symbols are coded as in a). The correlation is significant (p = 0.047). d) Mean visual cortex voxel (VC) NAA and the significance of group differences was calculated for optic neuritis groups as in b). The asterisk indicates statistically significant (p = 0.046) group differences. Error bars represent 2× standard error of the mean (SEM). e) RNFLT averages are shown in relation to corresponding NAA concentrations in normal-appearing white matter (NAWM). The symbols are coded as in a). No significant correlation was found (p = 0.531). f) Mean NAA in normal-appearing white matter (NAWM) and the significance of group differences was calculated for optic neuritis groups as in b) (p = 0.429). Error bars represent 2× standard error of the mean (SEM).</p

Statistical data for GEE and combined model GEE (NAWM = normal-appearing white matter, VC = visual cortex).

<p>Statistical data for GEE and combined model GEE (NAWM = normal-appearing white matter, VC = visual cortex).</p