BackgroundTeriflunomide, an inhibitor of dihydroorotate dehydrogenase, is thought to ameliorate multiple sclerosis by reducing activation-induced proliferation of lymphocytes, which is highly dependent on de novo pyrimidine synthesis. Nevertheless, its immunomodulatory effects on resident glial cells in the central nervous system are only poorly understood.MethodsIn this study, we employed physiologically relevant concentrations of teriflunomide and investigated its effects on survival, proliferation, activation, and function of primary rat microglia in vitro.ResultsWe demonstrate that teriflunomide had no cytotoxic effect on microglia and had only a minor impact on microglial activation. In a concentration- and time-dependent manner, teriflunomide significantly downregulated surface expression of the co-stimulatory molecule CD86. Furthermore, in the highest concentration applied (5 μM), it slightly increased the expression of interleukin-10 in microglia in response to lipopolysaccharide. Treatment with low concentrations of teriflunomide (0.25–1 μM) did not have any impact on the activation or proliferation of microglia. At 5 μM concentration of teriflunomide, we observed a reduction of approximately 30 % in proliferation of microglia in mixed glial cell cultures.ConclusionsTaken together, our in vitro findings suggest that at higher concentrations, teriflunomide potentially exerts its effects by reducing microglial proliferation and not by modulating the M1-/M2-like cell differentiation of primary rat microglia. Thus, teriflunomide has no major impact on the plasticity of microglia; however, the anti-proliferative and minimal anti-inflammatory effects might be clinically relevant for immune modulation in the treatment of neuroinflammatory CNS diseases such as multiple sclerosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0715-3) contains supplementary material, which is available to authorized users.

Remyelination is the natural repair mechanism in demyelinating disorders of the central nervous system (CNS) such as multiple sclerosis. Several animal models have been used to study demyelination and remyelination. Among toxic animal models, oral administration of the toxin cuprizone leads to white and gray matter demyelination. In contrast, focal demyelination models include the stereotactic application of a toxin such as lysolecithin or ethidium bromide. The injection procedure generates a local disruption of the blood-brain barrier (BBB) and might thus trigger a local inflammatory reaction and consequently may influence demyelination and remyelination. In order to study such consequences, we applied stereotactic injections in the cuprizone model where demyelination and remyelination are mediated independent of this procedure. Immunohistochemistry was performed to detect the presence of lymphocytes and activated glial cells in the injection area. Blood protein stainings were used to assess the integrity of the BBB and myelin staining to evaluate demyelination and remyelination processes. Stereotactic injection led to a local disruption of the BBB as shown by local extravasation of blood proteins. Along the injection canal, T and B lymphocytes could be detected and there was a tendency of a higher microgliosis and astrocytosis. However, these changes did not influence demyelination and remyelination processes at the site of injection, in the corpus callosum, or in the cerebral cortex. Our results suggest that a local stereotactic injection has no major impact on CNS demyelination and remyelination.

In vitro amyloid formation has been suggested to be a common property of any polypeptide chain depending on particular environmental conditions although in vivo amyloid fibril formation can be promoted by point mutations or triplet expansions. Here, we explored the influence of agitation on fibril formation of amyloidogenic alanine segments fused to Cold Shock Protein B (CspB) of Bacillus subtilis. While without agitation fibril formation was clearly dependent on the presence of an amyloidogenic alanine segment, fibril formation was independent of the amyloidogenic segment under agitation. Agitation even led to fibrillation of native CspB lacking the amyloidogenic segment. Furthermore, agitation not only influenced the kinetics of fibril formation, but also resulted in completely different fibril morphologies. These results indicate that experimental conditions can alter the region that undergoes a conformational change during in vitro fibrillation. Moreover, the data show that deductions from in vitro assays on in vivo fibril formation mechanisms are afflicted with a certain degree of uncertainty and therefore need to be cautiously discussed.

SummaryMultiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease triggered by infiltration of activated T cells into the central nervous system. Interferon (IFN)-b is an established, safe and effective treatment for patients with relapsing-remitting MS (RRMS). The cytokine can inhibit leucocyte infiltration into the central nervous system; however, little is known about the precise molecular mechanisms. Previously, in vitro application of IFN-b1b was shown to reduce CXCL12/ CXCR4-mediated monocyte migration. Here, we analysed the effects of IFN-b1b on CXCR4-dependent T cell function. In vitro exposure to IFN-b1b (1000 U/ml) for 20 h reduced CXCR4-dependent chemotaxis of primary human T cells from healthy individuals and patients with RRMS. Investigating the IFN-b1b/CXCR4 signalling pathways, we found no difference in phosphorylation of ZAP70, ERK1/2 and AKT despite an early induction of the negative regulator of G-protein signalling, RGS1 by IFNb1b. However, CXCR4 surface expression was reduced. Quantitative real time-PCR revealed a similar reduction in CXCR4-mRNA, and the requirement of several hours' exposure to IFN-b1b supports a transcriptional regulation. Interestingly, T cells from MS patients showed a lower CXCR4 expression than T cells from healthy controls, which was not reduced further in patients under IFN-b1b therapy. Furthermore, we observed no change in CXCL12-dependent chemotaxis in RRMS patients. Our results demonstrate clearly that IFN-b1b can impair the functional response to CXCR4 by down-regulating its expression, but also points to the complex in vivo effects of IFN-b1b therapy.

No abstract