Discussion

We show in the present study that patients with MS at different stages have reduced serum biopterin levels and temporarily increased neopterin concentrations suggesting high turnover and consumption, which is supported by a drop of biopterin ex vivo upon immune stimulation of human whole blood. Previous studies show that BH4 assists in NRF2 (nuclear factor erythroid 2-related factor 2) activation (Gangula et al., 2018; McNeill et al., 2015), which is a key mechanism of recently used MS drugs, fumaric acid esters (Linker et al., 2011). Hence, sapropterin supplementation might foster NRF2 with beneficial clinical outcome. However, we found that oral sapropterin (BH4, Kuvan®) treatment in mice mildly aggravated immunization-evoked EAE and increased the numbers of infiltrating T-cells in the spinal cord, without effect on overall numbers in blood and spleen. The EAE results suggest that sapropterin facilitated the invasion of T-cells into the CNS, via effects on the BBB rather than direct effects on T-cells. Sapropterin medication is safe and well tolerated in phenylketonuria patients (Feillet et al., 2008; Howells et al., 1986) and did not increase T-cell proliferation, but possibly needs to be used with caution in patients with autoimmunity. Although mechanistically different, this conclusion is supported by a previous study showing that BH4 deficient T-cells have a proliferation defect (Cronin et al., 2018), owing to a defect of mitochondrial iron transport and cytochrome C functions (Cronin et al., 2018).
Mechanistically we focused on systemic alterations of lipid signaling molecules and metabolic lipids rather than iron-mediated direct effects on T-cells because we have previously observed lipid alterations in a colitis model in dependence of BH4 (Zschiebsch et al., 2016) and AGMO is abundant in epithelial barriers. Epidemiology studies suggest beneficial effects of PUFAs in MS (Bjørnevik et al., 2017), in particular linolenic acid, which was neuroprotective in EAE mice (Bittner et al., 2013), and is generated via cytochrome B5 dependent stearoyl CoA desaturases and other fatty acid desaturases.
Indeed, gene expression analysis of spinal cord from EAE versus naïve mice revealed reduced levels of fatty acid desaturases and stearoyl-CoA desaturase (FADS1, SCD1 and SCD2) in EAE mice. SCD mediated desaturation is carried out with help of cytochrome B5 and is dependent on iron cycling. Like AGMO, it is a transmembrane ER enzyme that generates linolenic acid (FA18:3) from linoleic acid (FA18:2) (among others), hence linking iron, BH4 and lipid metabolism. Incorporation of PUFAs into biological barriers increases membrane fluidity and facilitates the insertion of receptors (Das et al., 2003).
Mice treated with sapropterin had lower plasma levels of linolenic acid, which plays an important role in BBB maintenance in the EAE model (Bittner et al., 2009) and has been suggested as supportive diet in MS (Bjornevik et al., 2019). In addition, sapropterin therapy in EAE was associated with an increase of plasma ceramides, which have been previously associated with the severity of EAE (Eberle et al., 2014; Schiffmann et al., 2012) suggesting that these changes are a biological correlate of the sapropterin-caused aggravation of the disease. They are not necessarily direct effects of BH4 on ceramide metabolism. Overall, the lipid alterations and clear discrimination of treatment groups based on lipids appear to be too strong to be mechanistically not linked with sapropterin. Lipid alterations may arise from changes of BH4-cofactor availability for the ER-localized lipid-metabolizing enzyme, AGMO. We observed high expression of AGMO in a LacZ reporter mouse at the brain-to-CSF barrier in the ependymal epithelium (Suppl. Fig 4), whereas endothelial NOS is highly expressed in brain endothelial cells and important for BBB functions (Wu et al., 2009). Hence, BH4 may act at two crucial barrier sites in the CNS and result in a permissive effect on immune cell invasion of the CNS. It is of note that oral sapropterin treatment in mice did not have such permissive effects on T-cell infiltration of the lamina propria in dextran sulfate sodium (DSS) evoked colitis model (Zschiebsch et al., 2016). The intestinal epithelial barrier and BBB differ in microenvironments, mesodermal versus endodermal origin and the molecular composition of the tight junctions (Daneman et al., 2009) and likely lipid composition of the membranes, which however has not yet been directly compared. Omega-3 lipids are protective at both sites (Bjornevik et al., 2019; Zhao et al., 2015) and high ceramides are detrimental at both sites (Oertel et al., 2017; Schiffmann et al., 2012). We hypothesize that the differences rely in the model per se. EAE is autoimmune driven whereas DSS disrupts the mucous layer and gives microbiota access to the intestinal wall. Oral sapropterin might also directly affect the gut microbiome. It is important that sapropterin treatment did not increase T-cell proliferation. Hence, it was not a general ”immune boost”. We infer that oral sapropterin is safe as supplementation, albeit possibly with caution with autoimmune-directed CNS disease.