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\begin{document}

\title{Cladribine modifies functional properties of microglia}



\author[1]{Line Jorgensen}%
\author[1]{Kirsten Hyrlov}%
\author[1]{Maria Elkjaer}%
\author[1]{Anna Weber}%
\author[2]{Anders Petersen}%
\author[3]{Asa Svenningsen}%
\author[1]{Zsolt Illes}%
\affil[1]{Odense University Hospital}%
\affil[2]{University of Copenhagen}%
\affil[3]{University of Southern Denmark}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background: Cladribine (CdA), an oral prodrug approved for the treatment
of relapsing multiple sclerosis, selectively depletes lymphocytes. CdA
passes the blood-brain barrier suggesting a potential effect on CNS
resident cells. Objective: We examined, if CdA modifies the phenotype
and function of na\selectlanguage{ngerman}ïve and activated primary mouse microglia, when
applied in different concentrations including 0.1-1 \selectlanguage{greek}µ\selectlanguage{english}M that putatively
overlaps human CSF concentrations. Methods: Primary microglia cultures
without stimulation or in the presence of proinflammatory
lipopolysaccharide (LPS) or anti-inflammatory IL-4 were co-treated with
different concentrations of CdA for 24 hours. Viability was assessed by
MTT assay. Phagocytotic ability and morphology were examined by flow
cytometry, and random migration by IncuCyte Zoom and TrackMate. Change
in gene expression was examined by qPCR, and protein secretion by Meso
Scale Discovery. Results: LPS and IL-4 upregulated deoxycytidine kinase
(DCK) expression. Only activated microglia were affected by CdA, and
this was unrelated to viability. CdA 0.1-1 \selectlanguage{greek}µ\selectlanguage{english}M significantly reduced
granularity, phagocytotic ability and random migration of activated
microglia. CdA 10 \selectlanguage{greek}µ\selectlanguage{english}M increased the IL-4-induced gene expression of Arg1
and LPS-induced expression of IL-1beta, TNF, iNOS, and Arg1, but protein
secretion remained unaffected. CdA 10 \selectlanguage{greek}µ\selectlanguage{english}M potentiated the increased
expression of anti-inflammatory TNFR2 but not TNFR1 induced by LPS.
Conclusion: Microglia acquire a less activated phenotype when treated
with 0.1--1 \selectlanguage{greek}µ\selectlanguage{english}M CdA that putatively overlaps human CSF concentrations.
This may be related to the upregulated gene expression of DCK upon
activation and suggests a potential alternative mechanism of CdA with
direct effect on CNS resident cells.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Cladribine (CdA), also known as 2-chlorodeoxyadenosine, is a synthetic
deoxyadenosine analogue that selectively depletes lymphocytes (1-3). CdA
is a prodrug, and accumulation of the active drug CdA triphosphate
depends on the ratio of deoxycytidine kinase (DCK), that catalyzes the
first of three phosphorylation steps, and 5'-nucleotidases (5'-NTases),
that converts phosphorylated cladribine to cladribine nucleoside. In
lymphocytes due to a high DCK to 5'-NTase ratio, accumulation of CdA
triphosphate results in apoptosis by several mechanisms (2, 3).

Cladribine tablets has been approved for the treatment of relapsing MS,
based in part on a placebo-controlled, double-blind, multicenter
phase-III trial (CLARITY). 3.5 mg/kg oral CdA reduced the annual relapse
rate by around 57\% (4), and the relative reduction in the risk of
3-month sustained progression of disability was 33\% compared to
placebo. The mean number of active T2 lesions on MRI was reduced by
73.4\% (4). The extension of the phase-III trial showed a durable effect
on MRI and clinical outcomes (5, 6).

The dose of 3.5 mg/kg CdA applied orally in short treatment cycles
causes a short-term lymphocyte depletion followed by
immune-reconstitution (7). However, CdA passes the blood-brain barrier
(BBB), and gives rise to 25\% of the plasma concentration in CSF
resulting in a maximum CSF concentration of 0.019-0.025 \selectlanguage{greek}µ\selectlanguage{english}M (2, 3), and
this may suggest a potential effect on CNS resident cells as well.

Microglia comprise 10-15\% of all glial cells in the CNS (8).
Pro-inflammatory microglia secrete pro-inflammatory cytokines,
chemokines, reactive oxygen and nitrogen species, which are toxic for
oligodendrocytes and neurons (9, 10). Lipopolysaccharides (LPS) induce a
pro-inflammatory phenotype of microglia characterized by the expression
of interleukin-1\selectlanguage{greek}β \selectlanguage{english}(IL-1\selectlanguage{greek}β\selectlanguage{english}), IL-6, major histocompatibility complex II
(MHC II), tumor necrosis factor (TNF) and inducible nitric oxide
synthase (iNOS) (11, 12). Microglia also exert neuroprotective and
repairing functions, and can produce neurotrophic factors and
anti-inflammatory cytokines (11, 13, 14). The anti-inflammatory cytokine
IL-4 induces the anti-inflammatory phenotype characterized by arginase 1
(Arg1) and IL-10 expression among others (11, 12, 15).

Data about the effect of CdA on microglia are scarce (16). When
investigating the effect of the drug in murine cells, higher CdA
concentrations are required because the activity of DCK is ten times
lower in mice than in humans (17, 18). Here, we examined the effect of
CdA on the phenotype and function of na\selectlanguage{ngerman}ïve and activated mouse microglia
in concentrations that putatively overlap with the levels in the human
CSF.

\textbf{Materials and Methods}

Animals

C57BL/6 mice were obtained from Taconic Ltd. (Ry, Denmark). Mice were
bred at the Biomedical Laboratory, University of Southern Denmark,
according to protocols and guidelines approved by the Danish Animal
Experiments Inspectorate (approval NO: 2014-15-00369). Animal
experiments complied with the EU Directive 2010/63/EU for animal
experiments.

Microglia cell culture

Mixed primary glial cell cultures were prepared by enzymatic
dissociation of postnatal P0-P5 mouse brains using Neural Tissue
Dissociation Kit (P) (Miltenyi Biotec, 130-092-628). The cells were
allowed to proliferate for 10 days prior to isolation of microglia by
the ``shake off'' method (19). The isolated microglia were on average
95\% pure, when characterized as
CD11b\textsuperscript{+}/CD45\textsuperscript{low} on the flow cytometer
(BD\textsuperscript{TM} LSR II, BD biosciences).

\emph{In vitro} \textbf{\emph{stimulation of microglia}}

CdA powder was provided by Merck. CdA was dissolved in dimethyl
sulfoxide (DMSO) (Sigma-Aldrich, D2438). Microglia were treated with
0.2\% DMSO, LPS (10 ng/ml) (Sigma-Aldrich, L2630), DMSO and LPS, one of
four concentrations of CdA (0.01 \selectlanguage{greek}µ\selectlanguage{english}M, 0.1 \selectlanguage{greek}µ\selectlanguage{english}M, 1 \selectlanguage{greek}µ\selectlanguage{english}M and 10 \selectlanguage{greek}µ\selectlanguage{english}M) alone or in
combination with LPS for 24 hours. For migration, the cells were
stimulated immediately before placement in the IncuCyte. Microglia were
stimulated with IL-4 (20 ng/ml) or LPS (10 ng/ml) alone or together with
CdA for 24 hours for qPCR and Meso Scale.

MTT viability assay

MTT solution (0.5 mg/ml) (Sigma-Aldrich, M0283) was added to LPS- and
CdA-pretreated microglia for 4 hours. The absorbance was measured by a
microplate reader (Molecular Devices, 6465).

Phagocytosis assay, size and granularity

Microglia were incubated with fluorescent latex beads and analyzed by
flow cytometry as described (20). Fluorescent latex beads (Polysciences,
Inc., 17154-10) were added to LPS and CdA treated microglia for 40 min
at 37\selectlanguage{ngerman}°C (sample) and 4°C (control). Cytochalasin D (5 \selectlanguage{greek}µ\selectlanguage{english}g/ml) was applied
prior to addition of the beads as negative control. Phagocytosis was
stopped by placing the cells on ice. Cells were detached by using 0.2\%
Trypsin-EDTA and analyzed by flow cytometry. Microglia size and
granularity were assessed by flow cytometry by measuring forward scatter
(FSC) and side scatter (SSC), respectively.

\par\null

Random migration assay

Microglia were seeded at a density of 12,000 cells/well in a PDL coated
96-well ImageLock Plate (Essen Bioscience, 4379) and stained with
CellTracker Red CMTPX Fluorescent Probe (4.5 \selectlanguage{greek}µ\selectlanguage{english}M) (Life Technologies).
DMSO (0.2\%), LPS (10 ng/ml), and CdA in different concentrations alone
(0.1-10 \selectlanguage{greek}µ\selectlanguage{english}M) or together with LPS were added, and cells were placed in
the IncuCyte Zoom live cell imaging system (Essen BioScience, USA).
Pictures were taken every 20 min for 24 hours. Single cell motility was
quantified using the Fiji plugin TrackMate for semi-automated particle
tracking (21). To detect individual cells in TrackMate, the Laplacian of
Gaussian (LoG) detector with estimate spot diameter of 24.12 \selectlanguage{greek}μ\selectlanguage{english}m and a
threshold of 0.3 was used. The simple linear assignment problem (LAP)
tracker with a linking maximum distance of 60 \selectlanguage{greek}µ\selectlanguage{english}m, a gap-closing max
distance of 15 \selectlanguage{greek}µ\selectlanguage{english}m and a gap closing max distance of 2 was used to track
cell migration through the time course movies. The number of spots in
track was set to 71.09 to exclude cells, which were not detectable
throughout the movies from the analysis.

RNA extraction and quantitative PCR

RNA was extracted using RNeasy micro kit (Qiagen, Denmark) and reverse
transcribed using High Capacity cDNA Reverse Transcription kit (Applied
Biosystems, 4374966). Quantitative PCR (qPCR) was performed on Bio-Rad
CFX Connect\textsuperscript{TM} Real-Time System (Software Bio-Rad CFX
Manager 3.1) using SYBR green chemistry (Thermo Scientific) and
corresponding primers (\textbf{Supplementary Table 1} ). The expression
levels are reported relative to the geometric mean of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine
phosphoribosyltransferase 1 (HPRT1).

Meso Scale Discovery (MSD) multiplex analysis

Cytokine levels in the microglia cell culture media were measured with
the Meso Scale Discovery (MSD, USA) proinflammatory mouse V-Plex Plus
kit (22).

Statistics

The statistical analyses were performed in Prism 6.01 (GraphPad) using
one-way ANOVA followed by Sidak's or Dunnett's multiple comparisons
tests or by unpaired T test with Welch's correction. Outliers were
removed using the ROUT method (Q=1\%). Differences among means with
p\textless{}0.05 were accepted as statistically significant, while
differences among means with 0.05\textless{}p\textless{}0.10 were
accepted as representing tendencies of difference.

\textbf{Results}

\selectlanguage{greek}\textbf{Cladribine (0.01-10 µM) does not influence the viability of
naïve or LPS-stimulated microglia}\selectlanguage{ngerman}

Microglia were treated with DMSO, LPS, DMSO and LPS, different
concentrations of CdA, and CdA together with LPS for 24 hours before
incubation with MTT. DMSO, LPS, and their combination did not affect the
viability of microglia compared to control (\textbf{Figure 1} ). CdA in
the applied concentration range (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M) did not affect the
viability of na\selectlanguage{ngerman}ïve and LPS-stimulated microglia either compared to DMSO
control (\textbf{Figure 1} ).

\selectlanguage{greek}\textbf{Cladribine (0.1 µM) decreases the granularity of LPS-stimulated
microglia}\selectlanguage{english}

The size and granularity of na\selectlanguage{ngerman}ïve and LPS-stimulated microglia treated
with CdA were investigated by flow cytometry (\textbf{Figure 2} ). LPS
stimulation increased the SSC mean fluorescent intensity (MFI)
indicating increased cellular granularity (\textbf{Figure 2A and B} ).
We observed a trend towards reduced granularity of LPS-stimulated
microglia treated with 0.1 \selectlanguage{greek}µ\selectlanguage{english}M CdA (p=0.07), while CdA did not influence
the granularity of na\selectlanguage{ngerman}ïve microglia (\textbf{Figure 2A and B} ). No
apparent difference in cell size reflected by FSC MFI was observed
(\textbf{Figure 2A and C} ).

\selectlanguage{greek}\textbf{Cladribine (0.1-1 µM) decreases the phagocytotic ability of
LPS-stimulated microglia}\selectlanguage{english}

To investigate the effect of CdA on phagocytosis by na\selectlanguage{ngerman}ïve and
LPS-stimulated microglia, the cells were incubated with fluorescent
latex beads, and analyzed by flow cytometry. LPS stimulation
significantly increased phagocytosis compared to control, while
treatment with cytochalasin D, an inhibitor of actin polymerization,
reduced phagocytosis (\textbf{Figure 3A} ). CdA in the range of 0.1-1 \selectlanguage{greek}µ\selectlanguage{english}M
significantly decreased phagocytosis by LPS-stimulated microglia, while
treatment with CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M) did not affect phagocytosis by na\selectlanguage{ngerman}ïve
microglia compared to DMSO control (\textbf{Figure 3A} ).

\selectlanguage{greek}\textbf{Cladribine (0.1-1 µM) decreases the motility of LPS-stimulated
microglia}\selectlanguage{english}

Microglia motility after CdA treatment was measured by random migration:
track displacement refers to the bee-line distance the cells migrate in
24 hours, while the total distance travelled is the actual distance
covered by the cells. Microglia were stained with cell tracker and
treated with DMSO, LPS, DMSO and LPS, different concentrations of CdA,
or CdA together with LPS. Movies were recorded, and the track
displacement and total distance travelled by the microglia were
determined.

The microglia track displacement was significantly increased by LPS
stimulation compared to untreated control (\textbf{Figure 3B} ).
Treatment with CdA 0.1 \selectlanguage{greek}µ\selectlanguage{english}M significantly decreased the track displacement
of LPS-stimulated microglia, whereas CdA (0.1-10 \selectlanguage{greek}µ\selectlanguage{english}M) did not alter the
track displacement of na\selectlanguage{ngerman}ïve microglia compared to DMSO control
(\textbf{Figure 3B} ).

The total distance travelled by microglia was not significantly
increased by LPS stimulation, though a trend was observed (p=0.07)
(\textbf{Figure 3C} ). CdA in the range of 0.1-1 \selectlanguage{greek}µ\selectlanguage{english}M significantly
decreased the total distance travelled by LPS-stimulated microglia
(\textbf{Figure 3C} ). CdA 0.1-10 \selectlanguage{greek}µ\selectlanguage{english}M did not have an effect on the total
distance travelled by na\selectlanguage{ngerman}ïve microglia compared to DMSO control
(\textbf{Figure 3C} ).

\selectlanguage{greek}\textbf{Cladribine (10 µM) alters expression but not secretion of pro-
and anti-inflammatory molecules by activated microglia}\selectlanguage{english}

Next, we investigated the effect of CdA on the relative mRNA expression
and secretion of selected pro- and anti-inflammatory markers after
stimulation with LPS or IL-4 for 24 hours. The expression of IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF,
IL-10, TNF-receptors, iNOS, and Arg1 were examined by qPCR. The
secretion of IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF, and IL-10 were investigated by Meso Scale
Discovery.

LPS stimulation resulted in an increased mRNA expression of IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF,
and IL-10 compared to control (\textbf{Figure 4A} ). Treatment with CdA
10 \selectlanguage{greek}µ\selectlanguage{english}M further increased the mRNA expression of IL-1\selectlanguage{greek}β \selectlanguage{english}and TNF in
LPS-stimulated cells (\textbf{Figure 4A} ). CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M) did not
affect the expression of these cytokines in na\selectlanguage{ngerman}ïve microglia (data not
shown).

The protein secretion of IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF, and IL-10 was increased by LPS
stimulation (\textbf{Figure 4B} ). In contrast, CdA (0.1-10 \selectlanguage{greek}µ\selectlanguage{english}M) did not
affect the secretion of IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF, and IL-10 from LPS-stimulated or
na\selectlanguage{ngerman}ïve microglia (\textbf{Figure 4B} ).

LPS stimulation increased the mRNA expression of both TNFR1 and TNFR2
compared to control. CdA 10 \selectlanguage{greek}µ\selectlanguage{english}M further increased the expression of TNFR2
but not TNFR1 (\textbf{Figure 5} ). CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M) did not affect the
expression of TNF receptors in na\selectlanguage{ngerman}ïve microglia (data not shown).

LPS stimulation resulted in a significantly increased expression of iNOS
and Arg1 mRNA compared to the untreated control, but these were reduced
by DMSO co-treatment (\textbf{Figure 6} ). Co-treatment with 10 \selectlanguage{greek}µ\selectlanguage{english}M CdA
significantly increased the expression of iNOS and Arg1 in
LPS-stimulated microglia compared to DMSO control (\textbf{Figure 6} ).
CdA did not affect the mRNA expression of iNOS or Arg1 in na\selectlanguage{ngerman}ïve
microglia (data not shown).

IL-4 stimulation for 24 hours only increased Arg1 expression, whereas no
changes were observed in mRNA expression of IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF, IL-10, TNFR1,
and TNFR2. Furthermore, the expression of these genes was not affected
by CdA co-treatment (\textbf{Figure 7 and} \textbf{Supplementary Figure
1-2} ). IL-4 did not induce the expression of iNOS (data not shown).
Similarly, no difference was observed in the secretion of IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF,
and IL-10 from IL-4-stimulated and CdA co-treated microglia
(\textbf{Supplementary Figure 1)} .

\textbf{LPS and IL-4 increases the mRNA expression of the enzyme DCK}

We examined the mRNA expression of DCK in na\selectlanguage{ngerman}ïve and activated microglia
treated with CdA. LPS stimulation increased the expression of DCK, while
IL-4 stimulation resulted in a similar trend compared to DMSO control
(p=0.08) (\textbf{Figure 8A and B} ). CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M) did not change
the increased expression induced by LPS and IL-4 (\textbf{Figure 8A and
B} ). The expression of DCK was not affected by CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M) in
na\selectlanguage{ngerman}ïve microglia either (data not shown).

\textbf{Discussion}

To determine the effect of CdA on murine microglia, we examined
phenotypic and functional properties of naïve, LPS-, and IL-4-stimulated
microglia treated with different concentrations of CdA for 24 hours. We
found that CdA only affects stimulated and not naïve microglia.
Furthermore, different concentrations of CdA influenced different
aspects of microglia function. CdA concentrations putatively overlapping
with CSF concentrations in humans (0.1-1 \selectlanguage{greek}µ\selectlanguage{english}M) significantly reduced the
granularity, phagocytotic ability, and motility of LPS-stimulated
microglia, while higher CdA concentration (10 \selectlanguage{greek}µ\selectlanguage{english}M) increased the mRNA
expression of pro- and anti-inflammatory molecules, but not protein
secretion. In addition, we found that activation of microglia increased
the expression of DCK responsible for the activation of the prodrug.

CdA can cross the BBB and thereby potentially exerts an effect on CNS
resident microglia (2, 3). The maximum plasma concentration of CdA after
administration of a 10 mg tablet is in the range of 0.07-0.1 \selectlanguage{greek}µ\selectlanguage{english}M (23). As
the CSF concentration is 25\% of the plasma concentration (2, 3), the
maximum CSF concentration attainable is approximately 0.019-0.025 \selectlanguage{greek}µ\selectlanguage{english}M.
The active form of CdA is CdA triphosphate, which is the phosphorylated
form of the drug (2, 3). The first step of this process is catalyzed by
deoxycytidine kinase (DCK), the activity of which is 10 times lower in
mice than humans (17, 18). The maximum CdA concentration detected in the
CSF of patients thus may correspond to murine concentrations in the
range of 0.1-1 \selectlanguage{greek}µ\selectlanguage{english}M. Therefore, we applied CdA concentrations of 0.01-10
\selectlanguage{greek}µ\selectlanguage{english}M in this study of murine microglia.

We first investigated whether the applied CdA concentrations were toxic
to microglia. We found that the viability of microglia was not affected
during the short culture conditions, similar to a previous study that
applied the intravenous compound to rat microglia cultures (16).
Therefore, we considered that using these concentrations for short time
stimulation would not have a depleting effect on microglia.

LPS stimulation resulted in a morphological change of microglia
characterized by an increase in SSC and thus a more granular morphology,
as expected. Co-treatment with CdA 0.1 \selectlanguage{greek}µ\selectlanguage{english}M showed a tendency towards
decreased granularity suggesting that CdA may have effect on the
functional properties of microglia.

Therefore, we examined the phagocytotic ability and motility of
microglia. We found that LPS stimulation significantly increased the
phagocytotic ability of microglia, which correlated with the
pro-inflammatory phenotype induced by LPS. Such increased phagocytotic
ability of LPS-stimulated microglia was decreased by CdA in the range of
0.1-1 \selectlanguage{greek}µ\selectlanguage{english}M. Furthermore, the decrease in phagocytosis by CdA corresponds
to the observed decreased granularity of microglia. CdA had no effect on
naive microglia, which is consistent with findings of former studies
performed on rat microglia (16), and murine dendritic cells (24).

Microglia motility is important for maintaining CNS homeostasis via
interaction with other cells and scanning of the CNS environment (8, 11,
25, 26). We measured the random migration by (i) track displacement,
i.e. the distance from start to end point, and (ii) total distance
travelled that includes twists and turns and describes the patrolling of
migrating cells. LPS stimulation significantly increased track
displacement and showed a tendency towards increased total distance
travelled. This is consistent with the acquisition of effector functions
induced by LPS and a tendency towards increased patrolling of the
surroundings. In contrast to random migration, LPS decreased chemotactic
migration of rat microglia (27). We also found that CdA (0.1-1 \selectlanguage{greek}µ\selectlanguage{english}M) did
not affect the motility of resting microglia.

Observing changes in morphology, phagocytotic ability and migration of
LPS-stimulated microglia, we next examined how CdA alters gene
expression and protein secretion of microglia stimulated with LPS and
with anti-inflammatory IL-4. LPS increased the expression of pro- and
anti-inflammatory markers consistent with a recently published study
(27). Co-treatment with CdA in the high concentration (10 \selectlanguage{greek}µ\selectlanguage{english}M) increased
the gene expression of both pro- and anti-inflammatory markers, such as
IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF, iNOS, Arg1, and TNFR2 suggesting a potentiating effect on
activated microglia. Interestingly, only the gene expression of
anti-inflammatory TNFR2, but not the pro-inflammatory TNFR1 (28) was
potentiated by CdA. The increased protein secretion of IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF, and
IL-10 induced by LPS was not further increased by CdA (0.1-10 \selectlanguage{greek}µ\selectlanguage{english}M)
consistent with a previous study on rat microglia (16). This suggests
that CdA only affects gene expression but not protein secretion. We may
speculate that accumulation of CdA in microglia may interfere with RNA
processing besides interference with DNA replication and this may alter
translational mechanisms.

We also found that IL-4 stimulation resulted in an increased gene
expression only of Arg1 but it was not affected by CdA co-treatment.
This could indicate that IL-4 is a less potent stimulus in the
short-term, and microglia may not be sufficiently activated for the
potentiating effect of CdA. In summary, CdA in the applied concentration
did not have effect on cytokine secretion of activated or resting
microglia in short-term primary cultures. The observed effect on gene
expression was achieved by higher concentrations of CdA than may be
expected in the human CSF during treatment.

Activation of microglia by LPS and IL-4 also increased the gene
expression of DCK that phosphorylates CdA to its active form. This may
suggest that activated microglia may increase its ability to respond to
CdA. In lymphocytes, CdA interferes with DNA synthesis and repair
resulting in depletion of both proliferating and resting cells (2, 3).
The mechanism by which CdA affects microglia functions remains to be
determined, and our data do not indicate microglia depletion in the
short term. A previous study also suggested that an additional mechanism
beside depletion may be responsible for the effects on microglia, such
as reduced proliferation (16). In dendritic cells, the function of CdA
is partially independent from its phosphorylation (24); but it may not
be true in microglia, as the effect of CdA on proliferation was lost
upon addition of a competitive substrate of DCK (16).

In summary, we found that CdA used in a concentration that may be
reached in the human CSF during approved treatment of MS, affected
activated but not na\selectlanguage{ngerman}ïve microglia; this may be related to the
upregulated gene expression of DCK upon activation. Co-treatment with
CdA reduced the phagocytotic ability and random migration of activated
microglia, while viability was not affected among the applied
conditions. Although gene expression of both pro- and anti-inflammatory
molecules was potentiated by CdA, it was induced only by higher
concentration than should be expected in the CSF, and protein secretion
was not altered. CdA also potentiated the gene expression of
anti-inflammatory TNFR2. These data suggest that beside depletion of
peripheral lymphocytes, CdA may induce additional effects particularly
in CNS resident cells that contribute to its beneficial effects in MS.

\textbf{Acknowledgements}

We thank Ulla Damgaard Munk for expert technical assistance.

\textbf{Funding:} Merck A/S, Denmark; an affiliate of Merck KGaA,
Darmstadt, Germany.

\textbf{Conflicts of interest:} LØJ received support for congress
participation from Merck. MLE received a speaker fee from Merck. AEP was
affiliated with Merck during conductance of the study, and AEP is now an
employee with Almirall, but the work is unrelated to this employment.
AEP also host a guest affiliation with University of Copenhagen. ZI has
served on scientific advisory boards, served as a consultant, received
support for congress participation, received speaker honoraria, and
received research support among others from Biogen, Merck-Serono,
Sanofi-Genzyme, Novartis, Roche, Lundbeckfonden. ÅFS, ABW, and KHH have
nothing to declare.

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\textbf{Figure legends}

\textbf{Figure 1}

\textbf{The effect of cladribine on the viability of naïve and
LPS-stimulated microglia}

Microglia were treated with DMSO (28.2 mM), LPS (10 ng/ml), DMSO and
LPS, different concentrations of CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M), and different
concentrations of CdA together with LPS for 24 hours, followed by an MTT
assay. The results (mean\selectlanguage{ngerman}±SD) are shown relative to DMSO control.

n=4-5 in each group, one-way ANOVA followed by multiple comparisons
tests.

\textbf{Figure 2}

\textbf{The effect of cladribine on microglia granularity and size}

Microglia were treated with DMSO (28.2 mM), LPS (10 ng/ml), DMSO and
LPS, different concentrations of CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M), and different
concentrations of CdA together with LPS for 24 hours. \textbf{A.} A
representative example of size (FSC) and granularity (SSC) of microglia
by flow cytometry. \textbf{B.} The effect of CdA on the granularity
(SSC) of na\selectlanguage{ngerman}ïve and LPS-stimulated microglia relative to DMSO control.
\textbf{C.} The effect of CdA on the size (FSC) of naïve and
LPS-stimulated microglia relative to DMSO control.

***p\selectlanguage{english}[?]0.001, n=3-4 in each group, one-way ANOVA followed by multiple
comparisons tests, mean+-SD.

\textbf{Figure 3}

\textbf{The effect of cladribine on the phagocytotic ability and random
migration of microglia}

\textbf{A.} Phagocytosis was quantified after microglia were treated
with DMSO (28.2 mM), LPS (10 ng/ml), DMSO and LPS, different
concentrations of CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M), and different concentrations of CdA
together with LPS for 24 hours. Fluorescent latex beads were added to
the cell media for 40 minutes. As negative control, microglia were
treated with cytochalasin D (5 \selectlanguage{greek}µ\selectlanguage{english}g/ml) for 30 minutes before addition of
latex beads. The cells were analyzed by flow cytometry, and the
phagocytotic ability was quantified by mean fluorescent intensity (MFI)
and presented relative to DMSO control. \textbf{B.} Track displacement
was quantified by analysis of timelapse videos using TrackMate after
microglia were treated with DMSO (28.2 mM), LPS (10 ng/ml), DMSO and
LPS, different concentrations of CdA (0.1-10 \selectlanguage{greek}µ\selectlanguage{english}M), and different
concentrations of CdA together with LPS for 24 hours. The results are
shown relative to DMSO control. \textbf{C.} Total distance travelled was
quantified by analysis of timelapse videos using TrackMate after
microglia were treated with DMSO (28.2 mM), LPS (10 ng/ml), DMSO and
LPS, different concentrations of CdA (0.1-10 \selectlanguage{greek}µ\selectlanguage{english}M), and different
concentrations of CdA together with LPS for 24 hours. The results are
shown relative to DMSO control.

*p[?]0.05, ****p[?]0.0001, n=3-5 in each group, one-way ANOVA followed by
multiple comparisons tests, mean\selectlanguage{ngerman}±SD.

\textbf{Figure 4}

\textbf{The effect of cladribine on the mRNA expression and protein
secretion of cytokines by LPS-stimulated microglia}

\textbf{A.} mRNA expression of IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF and IL-10 was quantified by
qPCR after microglia were treated with DMSO (28.2 mM), LPS (10 ng/ml),
DMSO and LPS, and different concentrations of CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M) together
with LPS for 24 hours. The expression is shown relative to DMSO
control.\textbf{B.} The protein concentration of IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF and IL-10 in
the microglia cell culture supernatant was quantified by Meso Scale. The
microglia were treated with DMSO (28.2 mM), DMSO and LPS, and different
concentrations of CdA (0.1-10 \selectlanguage{greek}µ\selectlanguage{english}M) together with LPS for 24 hours.

*p[?]0.05, **p[?]0.01, ***p[?] 0.001, ****p[?]0.0001, n=4 in each group,
unpaired t test with Welch's correction and one-way ANOVA followed by
Dunnett's multiple comparisons test, mean\selectlanguage{ngerman}±SD.

\textbf{Figure 5}

\textbf{The effect of cladribine on the mRNA expression of TNF receptors
in LPS-stimulated microglia}

mRNA expression of TNFR1 and TNFR2 was quantified by qPCR after
microglia were treated with DMSO (28.2 mM), LPS (10 ng/ml), DMSO and
LPS, and different concentrations of CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M) together with LPS
for 24 hours. The expression is shown relative to DMSO control.

*p[?]0.05, **p[?]0.01, ***p[?] 0.001, n=4 in each group, one-way ANOVA
followed by multiple comparisons tests, mean\selectlanguage{ngerman}±SD.

\textbf{Figure 6}

\textbf{The effect of cladribine on the mRNA expression of arginase 1
and iNOS in LPS-stimulated microglia}

mRNA expression of arginase 1 (Arg1) and iNOS was quantified by qPCR
after microglia were treated with DMSO (28.2 mM), LPS (10 ng/ml), DMSO
and LPS, and different concentrations of CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M) together with
LPS for 24 hours. The expression is shown relative to DMSO control.

**p[?]0.01, ***p[?] 0.001, n=4 in each group, one-way ANOVA followed by
multiple comparisons tests, mean\selectlanguage{ngerman}±SD.

\textbf{Figure 7}

\textbf{The effect of cladribine on the mRNA expression of arginase 1 in
IL-4-stimulated microglia}

mRNA expression of arginase 1 (Arg1) was quantified by qPCR after
microglia were treated with DMSO (28.2 mM), IL-4 (20 ng/ml), DMSO and
IL-4, and different concentrations of CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M) together with
IL-4 for 24 hours. The expression is shown relative to DMSO control.

*p[?]0.05, n=3-4 in each group, one-way ANOVA followed by multiple
comparisons tests, mean\selectlanguage{ngerman}±SD.

\textbf{Figure 8}

\textbf{The effect of cladribine on the mRNA expression of DCK in LPS-
and IL-4-stimulated microglia}

mRNA expression of deoxycytidine kinase (DCK) was quantified by qPCR and
is shown relative to DMSO control. \textbf{A.} Microglia were treated
with DMSO (28.2 mM), LPS (10 ng/ml), DMSO and LPS, and different
concentrations of CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M) together with LPS for 24
hours.\textbf{B.} Microglia were treated with DMSO (28.2 mM), IL-4 (20
ng/ml), DMSO and IL-4, and different concentrations of CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M)
together with IL-4 for 24 hours.

**p[?]0.01, ***p[?] 0.001, n=3-4 in each group, one-way ANOVA followed by
multiple comparisons tests, mean\selectlanguage{ngerman}±SD.

\textbf{Supplementary Figure 1}

\textbf{The effect of cladribine on the mRNA expression and protein
secretion of cytokines by IL-4-stimulated microglia}

\textbf{A.} mRNA expression of IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF and IL-10 was quantified by
qPCR after microglia were treated with DMSO (28.2 mM), IL-4 (20 ng/ml),
DMSO and IL-4, and different concentrations of CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M) together
with IL-4 for 24 hours. The expression is shown relative to DMSO
control. \textbf{B.} The protein concentration of IL-1\selectlanguage{greek}β, \selectlanguage{english}TNF and IL-10
in the microglia cell culture supernatant was quantified by Meso Scale.
Microglia were treated with DMSO (28.2 mM), DMSO and IL-4 (20 ng/ml),
and different concentrations of CdA (0.1-10 \selectlanguage{greek}µ\selectlanguage{english}M) together with IL-4 for
24 hours.

n=3-4 in each group, unpaired t test with Welch's correction and one-way
ANOVA followed by Dunnett's multiple comparisons test, mean\selectlanguage{ngerman}±SD.

\textbf{Supplementary Figure 2}

\textbf{The effect of cladribine on the mRNA expression of TNF receptors
in IL-4-stimulated microglia}

mRNA expression of TNFR1 and TNFR2 was quantified by qPCR after
microglia were treated with DMSO (28.2 mM), IL-4 (20 ng/ml), DMSO and
IL-4, and different concentrations of CdA (0.01-10 \selectlanguage{greek}µ\selectlanguage{english}M) together with
IL-4 for 24 hours. The expression is shown relative to DMSO control.

n=3-4 in each group, one-way ANOVA followed by multiple comparisons
tests, mean\selectlanguage{ngerman}±SD.\selectlanguage{english}
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