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.