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Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the progressive loss of oligodendrocytes and myelin and is associated with thalamic dysfunction. Cuprizone (CPZ)-induced general demyelination in rodents is a valuable model for studying different aspects of MS pathology. CPZ feeding is associated with the altered distribution and expression of different ion channels along neuronal somata and axons. However, it is largely unknown whether the copper chelator CPZ directly influences ion channels. Therefore, we assessed the effects of different divalent cations (copper; zinc) and trace metal chelators (EDTA; Tricine; the water-soluble derivative of CPZ, BiMPi) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that are major mediators of thalamic function and pathology. In addition, alterations of HCN channels induced by CPZ treatment and MS-related proinflammatory cytokines (IL-1beta; IL-6; INF-alpha; INF-beta) were characterized in C57Bl/6J mice. Thus, the hyperpolarization-activated inward current (I_h) was recorded in thalamocortical (TC) neurons and heterologous expression systems (mHCN2 expressing HEK cells; hHCN4 expressing oocytes). A number of electrophysiological characteristics of I_h (potential of half-maximal activation (V_0.5); current density; activation kinetics) were unchanged following the extracellular application of trace metals and divalent cation chelators to native neurons, cell cultures or oocytes. Mice were fed a diet containing 0.2% CPZ for 35 days, resulting in general demyelination in the brain. Withdrawal of CPZ from the diet resulted in rapid remyelination, the effects of which were assessed at three time points after stopping CPZ feeding (Day1, Day7, Day25). In TC neurons, I_h was decreased on Day1 and Day25 and revealed a transient increased availability on Day7. In addition, we challenged naive TC neurons with INF-alpha and IL-1beta. It was found that I_h parameters were differentially altered by the application of the two cytokines to thalamic cells, while IL-1beta increased the availability of HCN channels (depolarized V_0.5; increased current density) and the excitability of TC neurons (depolarized resting membrane potential (RMP); increased the number of action potentials (APs); produced a larger voltage sag; promoted higher input resistance; increased the number of burst spikes; hyperpolarized the AP threshold), INF-alpha mediated contrary effects. The effect of cytokine modulation on thalamic bursting was further assessed in horizontal slices and a computational model of slow thalamic oscillations. Here, IL-1beta and INF-alpha increased and reduced oscillatory bursting, respectively. We conclude that HCN channels are not directly modulated by trace metals and divalent cation chelators but are subject to modulation by different MS-related cytokines., Finanziert durch den Open-Access-Publikationsfonds der Westfälischen Wilhelms-Universität Münster (WWU Münster).

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.

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K2P18.1 is a relevant regulator. Here, we identify K2P18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-κB-mediated K2P18.1 upregulation in tTreg progenitors. K2P18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-κB- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K2P18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K2P18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K2P18.1 variant that is associated with poor clinical outcomes indicate that K2P18.1 also plays a role in human Treg development. Pharmacological modulation of K2P18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K2P18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K2P18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.

Expression of the Ca2+ activated potassium channel 3.1 (KCa3.1) channel (also known as the Gàrdos channel) is dysregulated in many tumor entities and has predictive power with respect to patient survival. Therefore, a positron emission tomography (PET) tracer targeting this ion channel could serve as a potential diagnostic tool by imaging the KCa3.1 channel in vivo. It was envisaged to synthesize [18F]senicapoc ([18F]1) since senicapoc (1) shows high affinity and excellent selectivity towards the KCa3.1 channels. Because problems occurred during 18F-fluorination, the [18F]fluoroethoxy senicapoc derivative [18F]28 was synthesized to generate an alternative PET tracer targeting the KCa3.1 channel. Inhibition of the KCa3.1 channel by 28 was confirmed by patch clamp experiments. In vitro stability in mouse and human serum was shown for 28. Furthermore, biodistribution experiments in wild type mice were performed. Since [18F]fluoride was detected in vivo after application of [18F]28, an in vitro metabolism study was conducted. A potential degradation route of fluoroethoxy derivatives in vivo was found which in general should be taken into account when designing new PET tracers for different targets with a [18F]fluoroethoxy moiety as well as when using the popular prosthetic group [18F]fluoroethyl tosylate for the alkylation of phenols., Finanziert durch den Open-Access-Publikationsfonds der Westfälischen Wilhelms-Universität Münster (WWU Münster).

Aims Thoracic aortic calcification (TAC) is measured by computed tomography (CT). We investigated the association of TAC-progression with incident cardiovascular (CV) events and all-cause mortality in a population-based cohort and to determine its predictive value for these endpoints. Methods and results In 3080 participants (45-74 years, 53.6% women), risk factors and TAC via CT were measured at baseline and at a second examination after 5.1 ± 0.3 years. Hard coronary, hard CV events as well as CV events including revascularization and all-cause mortality were recorded during a follow-up time of 7.8 ± 2.2 years after the second CT scan. Cox regression analysis determined the association of TAC-progression with observed endpoints. The predictive value of TAC-progression was assessed using Harrell's C index. We observed 81 hard coronary, 154 hard CV, 231 CV events including revascularization, and 266 deaths. In the crude analysis, event rates increased continuously with the level of TAC-change over 5 years for all endpoints. After adjustment, the significant association of TAC-progression with hard CV events [hazard ratio (HR) 1.28, 95% confidence interval (CI) 1.05-1.57] and all-cause mortality (HR 1.34, 95% CI 1.14-1.58) persisted, per one standard deviation increase in TAC-progression (log(TAC + 1)). Regarding aortic segments separately, HRs were consistently higher for descending thoracic aorta. When adding TAC (baseline and progression) to the model containing classical risk factors and coronary artery calcification (CAC), Harrell's C indices did not increase for any of the observed endpoints. Conclusion TAC-progression is associated with incident hard CV events and all-cause mortality but fails to improve event prediction over CAC.