Search

Additional file 2: Figure S2. Titration curves of oral cladribine treatment in mice. In preliminary experiments, we analyzed the plasma [ng/ml] and brain concentrations [ng/g] of oral cladribine treatment with different doses (3.25, 5 and 10 mg/kg bodyweight) over a time period of 2.5 h. Every 30 min blood was drawn and a proportion of animals was killed to obtain brain tissue for titration analyses. Curves show the maximal concentration of cladribine in blood and brain tissue (Cmax [ng/ml or ng/g]), the time to maximal concentration (tmax [hours]) and the area under the curve (AUC [ng*h/ml or ng*h/g]).

Additional file 3: Figure S3. Gating strategy for flow cytometry. Single immune cells from the periphery (A, here exemplary shown with spleen tissue) and the central nervous system (CNS; B, here exemplary shown with spinal cord tissue) were simultaneously analyzed by flow cytometry. Total leukocytes were identified by forward scatter (FSC) and sideward scatter (SSC) and cell-doublets were removed by FSC width and FSC height gating. From these cells, we identified leukocytes subsets based on their surface marker expression: CD45R+ B cells; CD3+CD4+ T-helper cells, CD3+CD8+ cytotoxic T cells and CD4+CD8+ double positive T cells; CD3−NK1.1+ natural killer (NK) cells; CD11b+CD11c− monocytes and macrophages (M/M) and CD11b+CD11c+ dendritic cells. Regarding the CNS tissue, prior to discrimination of leukocyte subsets, we differentiated microglia cells (CD45med) from infiltrated leukocytes (CD45high).

Additional file 7: Figure S7. Proliferation assay of splenocytes from naïve wild-type mice, with and without cladribine treatment in vitro. A: Proliferated cells in % after cladribine treatment (with 0.1 or 1 μM cladribine) versus vehicle-treated cells. We compared the unstimulated to the stimulated setting (stimulated with 1 μg/mL anti-CD3 and 2 μg/mL anti-CD28 for 3 days) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). B: Mean fluorescence intensities (MFIs) of migration and activation markers in all viable stimulated cells upon vehicle- compared to cladribine-treatment (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). p > 0.05 = ns, p < 0.05 = *, p < 0.001 = ***, p < 0.0001 = ****.

Additional file 1: Figure S1. Experimental scheme. Active MOG EAE was induced as previously described by immunization (day 0) of C57BL/6J mice with MOG35–55 peptide, followed by pertussis toxin (PTX) injections (day 0 and day 2) (15). Mice were divided into two experimental groups: Group 1 received cladribine via oral gavage (10 mg/kg from day 5 to day 9), while group 2 received only the vehicle for the same period of time. On day 10 post-EAE induction, focal EAE lesions were generated by stereotactic injection of proinflammatory cytokines (interferon gamma (INF-γ) and tumor necrosis factor alpha (TNF-α) into the auditory cortex to induce cortical grey matter lesions. Experimental read-out (electrophysiological recordings, flow cytometric immunophenotyping, histology) was performed either on dmax (defining the day of maximal clinical deterioration) or on day 27 post-EAE induction (to assess the chronical EAE state).

Additional file 6: Figure S6. Migration assay of leukocytes in organ cultures of spleens from naïve wild-type mice, with and without cladribine treatment in vitro. A: Migration ratios (number of migrated cells: total cell count) of viable (live), CD4+ or CD8+ T cells and B cells upon cladribine treatment (vehicle (migration) versus cladribine 0.1 µM (migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs, p-values > 0.05). B: Flow cytometric analysis of the immune cell distribution in % in spleens (vehicle or cladribine (organ)) and the migrated immune cells (vehicle or cladribine (migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). C: Mean fluorescence intensities (MFIs) of migration and activation markers in viable CD4+ cells still located in the respective spleen (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). D: MFIs of migration and activation markers in viable CD8+ cells still located in the respective spleen (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs, p-values > 0.05). E: MFIs of migration and activation markers in viable B cells still located in the respective spleen (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). p > 0.05 = ns, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****.

Additional file 5: Figure S5. Migration assay of leukocytes in organ cultures of cervical LNs from naïve wild-type mice, with and without cladribine treatment in vitro. A: Migration ratios (number of migrated cells: total cell count) of viable (live), CD4+ or CD8+ T cells and B cells after cladribine treatment (vehicle (migration) versus cladribine 0.1 µM (migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs, p-values > 0.05). B: Flow cytometric analysis of the immune cell distribution in % in LNs (vehicle or cladribine (organ)) and the migrated immune cells (vehicle or cladribine (migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). C: Mean fluorescence intensities (MFIs) of migration and activation markers in viable CD4+ cells still located in the respective LN (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). D: MFIs of migration and activation markers in viable CD8+ cells still located in the respective LN (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs, p-values > 0.05). E: MFIs of migration and activation markers in viable B cells still located in the respective LN (vehicle or cladribine (organ)) compared to those after egress (vehicle or cladribine ((migration)) (n = 6 for both vehicle and cladribine treatment, two-way ANOVAs). p > 0.05 = ns, p < 0.05 = *, p < 0.01 = **, p < 0.0001 = ****.

Additional file 4: Figure S4. Gating strategy for sorting of macrophages. CD11b+ cells were isolated from murine CNS and spinal cord via magnetic beads. Subsequently, CD11b+ cells were simultaneously analyzed and sorted by flow cytometry. Total CD11b+ cells were identified by forward scatter (FSC) and sideward scatter (SSC) (A), and cell-doublets were removed by SSC width and SSC height gating (B). From these cells, we discriminated macrophages based on their surface marker expression of CD45highCD11bhigh (C). Macrophages were sorted for downstream experiments (D). Plots (E)-(F) show the distribution of microglia (CD45intermCD11bhigh) in comparison to macrophages.

Additional file 1:Supplementary Figure S1. Quantitative K2P5.1 expression on the protein level in cells from RA patients compared to healthy controls. Western blot analysis of five individual RA patients (one to five) compared to two healthy controls (HD). Respective DAS28 scores are indicated at the upper part of the figure. (PDF 102 KB)

Authors’ original file for figure 4

Additional file 5: Figure S5. The amyloid precursor protein (APP) accumulation upon de- and remyelination in frontal neocortical regions. Exemplary pictures show APP staining in the lower layers of frontal neocortical regions in control conditions (left), at 6 weeks after starting the cuprizone diet (cupri 6 weeks – full demyelination, middle), and at full remyelination 6 weeks after reintroduction of normal food (right). Note that an increase of positive cells, indicating an accumulation of APP in the neuronal soma, occurred slowly at the onset of the cuprizone diet to reach a significant threshold at remyelinating phases. On the right, bar graphs show the number of APP positive cells (cells/mm2) in Cx.

Additional file of Expression of K2P5.1 potassium channels on CD4+T lymphocytes correlates with disease activity in rheumatoid arthritis patients

Additional file of Expression of K2P5.1 potassium channels on CD4+T lymphocytes correlates with disease activity in rheumatoid arthritis patients

Additional file 6: Figure S6. Exploratory and grooming behavior were altered by cuprizone diet and its withdrawn. (a) Bar graph showing quantification of vertical exploratory behavior. Animals show a significant increase in comparison to control 2- and 6 weeks after the beginning of the diet. (b) Bar graph showing the quantification of grooming behavior. The latter is often considered a indicator of stress levels I rodents and here it is significantly decreased, in comparison to control, in almost all experimental groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Additional file 2: Figure S2. Representation of technical and biological replicates for histological evaluation of PLP intensity, astrocytosis and microglial activation in the Cx. (a) Scatter plots and graphs show the variability of the data acquired and used for histological evaluation on myelin intensity by using the specific marker PLP in the cortex (upper panel). (b) Scatter plots and graphs show the variability of the data acquired and used for histological evaluation of the number of astrocytes by using the specific marker GFAP in the cortex (mid panel). Simplified bar graphs are shown in the main figures.

Additional file 2:Supplementary Figure S2. Influence of therapeutic agents on K2P5.1 expression. A) Correlation between K2P5.1 expression levels (Δct values) on CD4+ T lymphocytes and DAS28 scores is shown for patients with convential treatments (black diamonds), TNFα inhibitors (grey diamonds) and rituximab therapy (white diamonds). B) Patient subgroups are shown with conventional treatment (left side), TNFα inhibitors (middle side) and rituximab (right side). C) The left bar graph representation shows the correlation coefficients between DAS28 and K2P5.1 expression levels. The DAS28 score for the treatment subgroups is shown on the right side. (PDF 63 KB)

Additional file 3: Figure S3. Structural and anatomical thalamic grey matter changes during de- and remyelination in the cuprizone model. (a) Exemplary pictures show staining for the specific myelin marker PLP in coronal mouse slices containing the ventrobasal complex of the thalamus (VB) in control conditions (left), at 6 weeks after starting the cuprizone diet (cupri 6 weeks – full demyelination, middle), and at full remyelination 6 weeks after reintroduction of normal food (right). Note the decreased signal for PLP indicating demyelination in the cupri 6 weeks group in comparison to control, and a persistent low PLP signal during remyelination. On the right, bar graphs show the quantification of myelin loss and regain for all groups and all investigated time points. (b) Exemplary pictures show staining for the specific astrocytic marker GFAP in coronal mouse slices containing the ventrobasal complex of the thalamus in control conditions (left), at 6 weeks after starting the cuprizone diet (cupri 6 weeks – full demyelination, middle), and at full remyelination 6 weeks after reintroduction of normal food (right). Note the increased number of astrocytes indicating astrocytosis in the cupri 6 weeks group in comparison to control and remy 6 weeks groups. On the right, bar graphs show the number of astrocytes (cells/mm2) in VB, this increased according to diet progression and cuprizone withdrawal.

Additional file 4: Figure S4. Representation of technical and biological replicates for histological evaluation of PLP intensity, astrocytosis and microglial activation in the thalamus. (a) Scatter plots and graphs show the variability of the data acquired and used for histological evaluation on myelin intensity by using the specific marker PLP in the thalamus (upper panel). (b) Scatter plots and graphs show the variability of the data acquired and used for histological evaluation of the number of astrocytes by using the specific marker GFAP in the thalamus (mid panel).

Additional file of Expression of K2P5.1 potassium channels on CD4+T lymphocytes correlates with disease activity in rheumatoid arthritis patients

Additional file of Expression of K2P5.1 potassium channels on CD4+T lymphocytes correlates with disease activity in rheumatoid arthritis patients

Additional file 1: Figure S1. Representation of technical and biological replicates for histological evaluation of PLP intensity, astrocytosis and microglial activation in the CC. (a) Scatter plots and graphs show the variability of the data acquired and used for histological evaluation on myelin intensity by using the specific marker PLP in the corpus callosum (upper panel). (b) Scatter plots and graphs show the variability of the data acquired and used for histological evaluation of the number of astrocytes by using the specific marker GFAP in the corpus callosum (mid panel). Simplified bar graphs are shown in the main figures.

Additional file of Expression of K2P5.1 potassium channels on CD4+T lymphocytes correlates with disease activity in rheumatoid arthritis patients

Authors’ original file for figure 2

Additional file of Expression of K2P5.1 potassium channels on CD4+T lymphocytes correlates with disease activity in rheumatoid arthritis patients

Authors’ original file for figure 1

Additional file of Expression of K2P5.1 potassium channels on CD4+T lymphocytes correlates with disease activity in rheumatoid arthritis patients

Authors’ original file for figure 3

Authors’ original file for figure 2

Authors’ original file for figure 3

Authors’ original file for figure 1

Authors’ original file for figure 4