Cannabidiol (CBD): a killer for inflammatory rheumatoid arthritis synovial fibroblasts

Abstract

Cannabidiol (CBD) is a non-intoxicating phytocannabinoid from cannabis sativa that has demonstrated anti-inflammatory effects in several inflammatory conditions including arthritis. However, CBD binds to several receptors and enzymes and, therefore, its mode of action remains elusive. In this study, we show that CBD increases intracellular calcium levels, reduces cell viability and IL-6/IL-8/MMP-3 production of rheumatoid arthritis synovial fibroblasts (RASF). These effects were pronounced under inflammatory conditions by activating transient receptor potential ankyrin (TRPA1), and by opening of the mitochondrial permeability transition pore. Changes in intracellular calcium and cell viability were determined by using the fluorescent dyes Cal-520/PoPo3 together with cell titer blue and the luminescent dye RealTime-glo. Cell-based impedance measurements were conducted with the XCELLigence system and TRPA1 protein was detected by flow cytometry. Cytokine production was evaluated by ELISA. CBD reduced cell viability, proliferation, and IL-6/IL-8 production of RASF. Moreover, CBD increased intracellular calcium and uptake of the cationic viability dye PoPo3 in RASF, which was enhanced by pre-treatment with TNF. Concomitant incubation of CBD with the TRPA1 antagonist A967079 but not the TRPV1 antagonist capsazepine reduced the effects of CBD on calcium and PoPo3 uptake. In addition, an inhibitor of the mitochondrial permeability transition pore, cyclosporin A, also blocked the effects of CBD on cell viability and IL-8 production. PoPo3 uptake was inhibited by the voltage-dependent anion-selective channel inhibitor DIDS and Decynium-22, an inhibitor for all organic cation transporter isoforms. CBD increases intracellular calcium levels, reduces cell viability, and IL-6/IL-8/MMP-3 production of RASF by activating TRPA1 and mitochondrial targets. This effect was enhanced by pre-treatment with TNF suggesting that CBD preferentially targets activated, pro-inflammatory RASF. Thus, CBD possesses anti-arthritic activity and might ameliorate arthritis via targeting synovial fibroblasts under inflammatory conditions.

Full text

Lowin et al. Cell Death and Disease (2020) 11:714
https://doi.org/10.1038/s41419-020-02892-1 Cell Death & Disease
ARTICLE Open Access
Cannabidiol (CBD): a killer for inflammatory
rheumatoid arthritis synovial fibroblasts
Torsten Lowin
1
, Ren Tingting
1
, Julia Zurmahr
1
, Tim Classen
2
, Matthias Schneider
1
and Georg Pongratz
1
Abstract
Cannabidiol (CBD) is a non-intoxicating phytocannabinoid from cannabis sativa that has demonstrated anti-
inflammatory effects in several inflammatory conditions including arthritis. However, CBD binds to several receptors
and enzymes and, therefore, its mode of action remains elusive. In this study, we show that CBD increases intracellular
calcium levels, reduces cell viability and IL-6/IL-8/MMP-3 production of rheumatoid arthritis synovial fibroblasts (RASF).
These effects were pronounced under inflammatory conditions by activating transient receptor potential ankyrin
(TRPA1), and by opening of the mitochondrial permeability transition pore. Changes in intracellular calcium and cell
viability were determined by using the fluorescent dyes Cal-520/PoPo3 together with cell titer blue and the
luminescent dye RealTime-glo. Cell-based impedance measurements were conducted with the XCELLigence system
and TRPA1 protein was detected by flow cytometry. Cytokine production was evaluated by ELISA. CBD reduced cell
viability, proliferation, and IL-6/IL-8 production of RASF. Moreover, CBD increased intracellular calcium and uptake of
the cationic viability dye PoPo3 in RASF, which was enhanced by pre-treatment with TNF. Concomitant incubation of
CBD with the TRPA1 antagonist A967079 but not the TRPV1 antagonist capsazepine reduced the effects of CBD on
calcium and PoPo3 uptake. In addition, an inhibitor of the mitochondrial permeability transition pore, cyclosporin A,
also blocked the effects of CBD on cell viability and IL-8 production. PoPo3 uptake was inhibited by the voltage-
dependent anion-selective channel inhibitor DIDS and Decynium-22, an inhibitor for all organic cation transporter
isoforms. CBD increases intracellular calcium levels, reduces cell viability, and IL-6/IL-8/MMP-3 production of RASF by
activating TRPA1 and mitochondrial targets. This effect was enhanced by pre-treatment with TNF suggesting that CBD
preferentially targets activated, pro-inflammatory RASF. Thus, CBD possesses anti-arthritic activity and might ameliorate
arthritis via targeting synovial fibroblasts under inflammatory conditions.
Introduction
Cannabidiol (CBD) is a non-intoxicating cannabinoid
found in cannabis sativa
1
. In contrast to the psychoactive
constituent tetrahydrocannabinol (THC), CBD demon-
strates no direct effect at cannabinoid receptors 1 and 2
(CB
1
and CB
2
) but modulates the effect of agonists sug-
gesting an allosteric function
2
. In addition, CBD binds to
PPARγ, GPR3/6/12/18/55, TRPV1/2, TRPA1, 5-
hydroxytryptamine receptor, and mitochondrial pro-
teins
3–11
. Despite its promiscuous pharmacology, CBD is
well tolerated even when given in high concentra-
tions
12,13
. Side effects of CBD in humans include diarrhea
and fatigue and, more importantly, CBD interacts with
other drugs since it is metabolized by CYP enzymes in the
liver thereby inhibiting the degradation of other ther-
apeutic compounds
14,15
. While the therapeutic benefits of
CBD in childhood epilepsy are well documented, its
effects on inflammation have only been investigated in
animal models
13,16
. Studies in rodents with osteoarthritis
or collagen-induced arthritis demonstrated anti-
inflammatory and analgesic effects of CBD, but these
studies did not identify the mechanism of action
17–19
.
© The Author(s) 2020
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, whi ch permits use, sharing, adaptation, distribution and reproduction
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Correspondence: Torsten Lowin (torsten.lowin@med.uni-duesseldorf.de)
1
Poliklinik, Funktionsbereich & Hiller Forschungszentrum für Rheumatologie,
University Hospital Duesseldorf, D-40225 Duesseldorf, Germany
2
Klinik für Orthopädie/Orthopädische Rheumatologie, St. Elisabeth-Hospital
Meerbusch-Lank, D-40668 Meerbusch, Germany
Edited by H.-U. Simon
Official journal of the Cell Death Differentiation Association
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Here, we investigate the effect of CBD on intracellular
calcium, cell viability, and cytokine production in rheu-
matoid arthritis synovial fibroblasts (RASF). RASF are one
major contributor of joint destruction in RA as they
secrete pro-inflammatory cytokines and matrix degrading
enzymes
20
. In fact, subsets of RASF selectively mediate
joint destruction or the inflammatory response, empha-
sizing their important role in the pathogenesis of RA
21
.In
previous studies, we already identified TRPA1 as a ther-
apeutic target since the TRPA1 agonist Polygodial selec-
tively deleted TNF-activated RASF
22
. CBD also binds
TRPA1
7,23
, and therefore we hypothesized that CBD has
detrimental effects on cell viability, which might explain
in part its mechanism of action at sites of inflammation.
Results
CBD reduces cell viability and proliferation of RASF
Overthecourseof6hwefoundthatCBD(≥5µM)
decreases cell viability (Fig. 1a, b), but a stimulatory effect was
detected for 1 µM CBD in TNF pre-incubated RASF (Fig.
1b). CBD combined with the TRPA1 antagonist, A967079,
recovered cell viability (Fig. 1h). TRPA1 is upregulated by
TNF (Fig. 1f) and we also detected an increase of TRPA1
mRNA by real-time PCR after 24 h under the influence of
TNF (data not shown). Ruthenium Red (RR) also reduced the
detrimental effects of CBD (Fig. 1i). Surprisingly, 4,4′-Dii-
sothiocyanatostilbene-2,2′-disulfonate (DIDS), supported cell
viability at low CBD concentrations but enhanced its cyto-
toxic effects at concentrations ≥1µM (Fig. 1j), while
Cyclosporin A (CsA), blocked the effects of CBD (Fig. 1k).
Since RealTime-Glo assays wereconductedat37°Cin
serum-free medium but without CO
2
and humidity control,
we also confirmed these results in cell titer blue endpoint
assays (Fig. 5d). In vivo, CBD is bound to lipoproteins/
albumin lowering the available concentration of free CBD
24
.
Therefore, we investigated the effect of fetal calf serum
content with CBD on proliferation. CBD in concentrations
≥5 µM reduced proliferation of RASF in medium without or
2% FCS (Fig. 1c, d). TNF pre-stimulation enhanced pro-
liferation at 5 µM CBD in 0% FCS (Fig. 1c) while the opposite
was true using 2% FCS (Fig. 1d). With 10% FCS, CBD
inhibited proliferation of RASF only at 20 µM (Fig. 1e).
DMSO (vehicle control) alone had a stimulatory effect on
proliferation (Fig. 1e, green line). These findings underline
the need for relatively high concentrations of CBD when
used in in vivo settings
13
.
CBD increases intracellular calcium and its effects are
enhanced by TNF
CBD influences calcium mobilization
9,23,25
and we
found that concentrations ≥5 µM increased intracellular
calcium (Fig. 2a). The potency of CBD was enhanced by
pre-incubation with TNF for 72 h (Fig. 2b). Under these
conditions, intracellular calcium levels were significantly
Fig. 1 Assessment of cell viability, TRPA1 expression, and RASF proliferation. a,bMean cell viability of unstimulated (a) or TNF pre-stimulated
(b) RASF after CBD challenge monitored in real-time over the course of 375 min. c–eMean proliferation of RASF with (red bars) and without (white
bars) TNF pre-stimulation in response to CBD in medium containing 0% FCS (c), 2% FCS (d), and 10% FCS. The dotted line represents the
unstimulated control, which was set to 100%. fFlow cytometric detection of TRPA1 protein in RASF with or without TNF stimulation for 72 h. g–k
Mean cell viability of TNF pre-stimulated RASF after CBD challenge and concomitant addition of inhibitors over the course of 20h. nis the number of
replicates and patient samples investigated. ANOVA with Dunnett’s T3 post-hoc test was used for comparisons in a,b,g–k. ANOVA with Bonferroni
post-hoc test was used for comparisons in c–e. Two-tailed t-test was used for comparisons in f.*p< 0.05; **p< 0.01, ***p< 0.001 vs control. The error
bars in c–frepresent the standard error of mean (sem).
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increased compared to untreated RASF (Fig. 2d). When
extracellular calcium was omitted by using PBS, CBD still
increased intracellular calcium although to a smaller
extent (Fig. 2c) Besides calcium, we also analyzed the
uptake of the cell viability dye PoPo3 iodide under CBD
stimulation. PoPo3 uptake increases when membrane
integrity is compromised during apoptosis or necrosis.
CBD dose-dependently increased the uptake of PoPo3
which was enhanced by extracellular calcium and TNF
pre-stimulation (Fig. 2f, g). Basal uptake of PoPo3 and
intracellular calcium levels were increased by TNF pre-
treatment (Supplementary Fig. 3A, B). The detrimental
effect of CBD on cell viability was also confirmed in the
XCELLigence system using untreated RASF (Supple-
mentary Fig. 1).
Calcium mobilization and PoPo3 uptake partly depend on
TRPA1 activation
Since CBD binds TRPV1, TRPV2, and TRPA1
7
we
investigated the involvement of these ion channels.
RR, a general inhibitor for several TRP channels
26–28
,
reduced intracellular calcium levels (Fig. 3e, f) and
PoPo3 uptake (Fig. 3g, h) but the magnitude of inhi-
biton was small. RR did not change basal calcium
levels or PoPo3 uptake in unstimulated but did so in
TNF pre-stimulated RASF (Supplementary Fig. 3).
Next, we combined CBD with the TRPV1 antagonist
Capsazepine (CPZ)
29
. CPZ had only a minor influence
on CBD-induced calcium levels and PoPo3 uptake
(Fig. 3i–l). Of note, CPZ also modulated basal calcium
and PoPo3 levels (Supplementary Fig. 3) With TRPA1
inhibition we found that without TNF pre-stimulation,
the antagonist A967079 (10 µM) increased intracel-
lular calcium (Fig. 3m) but decreased it when RASF
were pre-incubated with TNF (Fig. 3n). A967079
increased PoPo3 uptake under basal conditions
(Fig. 3o) but decreased it after TNF pre-incubation
(Fig. 3p). Furthermore, A967079 enhanced basal cal-
cium levels and PoPo3 uptake (Supplementary
Fig. 3A–D).
Fig. 2 Mean intracellular calcium levels and PoP3 uptake of RASF in response to CBD. a–cIntracellular calcium mobilization of RASF after CBD
challenge in HBSS (a,b) or without extracellular Ca
2+
(PBS; c). The EC
50
values obtained for the increase of intracellular calcium were significantly
different (p< 0.001) between unstimulated and TNF-pre-stimulated RASF. e–gPoPo3 uptake by RASF after CBD challenge in HBSS (e,f) or without
extracellular Ca
2+
(PBS; f). RASF were pre-stimulated with TNF (b,c,f,g) or untreated (a,e). d,hComparison between unstimulated and TNF pre-
treated RASF regarding intracellular calcium levels (d) and PoPo3 uptake (h). nis the number of experiment replicates from 29 different patient
samples (a,e), 38 patient samples (b,f), and 13 patient samples (c,g). ***p< 0.001, *p< 0.05 for differences between [c] of CBD. ANOVA with
Dunnett’s T3 post-hoc test was used for all comparisons.
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Mitochondrial targets mediate the effects of CBD
We investigated four proposed mitochondrial targets
for CBD: the mitochondrial calcium uniporter (MCU), the
sodium/calcium exchanger (NCLX)
30
, the voltage-gated
anion channel (VDAC1)
8
and the mitochondrial mem-
brane permeability transition pore (mPTP) which initiates
apoptotic and necrotic events
31
.While we detected a
minor influence of NCLX and MCU (Supplementary
results and Supplementary Fig. 2) on intracellular calcium
levels and PoPo3 uptake, inhibition of mPTP exerted the
strongest influence (Fig. 3q–t). CBD-induced changes in
intracellular calcium were reduced by the mPTP inhibitor
CsA (Fig. 3q). In TNF pre-stimulated RASF, CsA reduced
calcium levels over all CBD concentrations (Fig. 3r). In
addition, CsA accelerated PoPo3 uptake (Fig. 3s, t) but did
not alter basal calcium or PoPo3 levels (Supplementary
Fig. 3A–D). Next, we combined CBD with DIDS, which is
an inhibitor of VDAC in the outer mitochondrial and the
plasma membrane
32,33
. CBD stabilizes a closed con-
formation of VDAC, which excludes the exchange of
metabolites from the cytosol into mitochondria, but
enhances its calcium transport function
8,34
. DIDS
increased CBD-induced calcium mobilization regardless
of TNF pre-stimulation (Fig. 3u, v) and it completely
abolished PoPo3 uptake under all conditions (Fig. 3w, x).
DIDS also increased basal calcium levels in TNF pre-
stimulated RASF (Supplementary Fig. 3B) and decreased
PoPo3 levels (Supplementary Fig. 3C, D). Since we found
TRPA1 to be involved in the effects of CBD (Figs. 1h and
3m–p), we were interested in the cellular localization of
this receptor. Since we assumed this to be an intracellular
site, we used Thapsigargin to deplete calcium stores in the
endoplasmatic reticulum (ER)
35,36
and Gly-Phe-
β-naphthylamide (GPN), which disrupts lysosomes and
releases calcium stored in this organelle
37,38
. We found
that GPN reduced the elevation of intracellular calcium by
CBD (p< 0.001, Fig. 4e, f), while PoPo3 uptake was sig-
nificantly enhanced (Fig. 4g, h). Of note, GPN per se
induced an increase of intracellular calcium and PoPo3
uptake (Supplementary Fig. 3A–D). Similarly, the inhi-
bitor of the endoplasmic reticulum Ca
2+
-ATPase, Thap-
sigargin, reduced the CBD-induced increase in
Fig. 3 Modulation of intracellular calcium levels and PoP3 uptake
of RASF after CBD challenge with concomitant inhibition of
membrane TRP channels and mitochondrial targets. Mean
intracellular calcium mobilization (a,b,e,f,i,j,m,n,q,r,u,v) and
mean PoPo3 uptake (c,d,g,h,k,l,o,p,s,t,w,x) of RASF after CBD
challenge and concomitant inhibition with the antagonists given in
the figure. nis the number of experiment replicates from 24 patient
samples (a,c), 33 patient samples (b,d), and 8 patient samples (n,p).
e–x(except n,p)nnumber equals number of replicates and different
patient samples. ANOVA with Dunnett’s T3 post-hoc test was used for
all comparisons versus (a–d). *p< 0.05, ***p< 0.001.
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Fig. 4 Modulation of intracellular calcium levels and PoP3 uptake of RASF by CBD with concomitant manipulation of lysosomal and
endoplasmatic reticulum calcium stores or inhibition of organic cation transport. Mean intracellular calcium mobilization (a,b,e,f,i,j,m,n)
and mean PoPo3 uptake (c,d,g,h,k,l,o,p) of RASF after CBD challenge and concomitant inhibition with the antagonists given in the figure. ANOVA
with Dunnett’s T3 post-hoc test was used for all comparisons. ***p< 0.001.
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intracellular calcium levels (Fig. 4i, j) and slightly atte-
nuated PoPo3 uptake (Fig. 4k, l) but increased basal cal-
cium and PoPo3 levels (Supplementary Fig. 3A–D).
Cationic and uncharged compounds are taken up by
organic cation transporters (OCT)
39,40
and therefore we
assessed the effects of Decynium-22 (D22), an inhibitor of
all OCT isoforms on intracellular calcium and the uptake
of PoPo3. Under all conditions, we found that D22 pre-
vented the increase of intracellular calcium/PoPo3 uptake
induced by CBD (Fig. 4m–p) and it reduced basal intra-
cellular calcium (Supplementary Fig. 3A, B) but slightly
elevated PoPo3 levels (Fig. 3c, d). Lastly, we determined
the influence of CBD on the production of IL-6, IL-8, and
MMP-3 by RASF. CBD (10 µM and 20 µM) significantly
decreased the production of IL-6 (Fig. 5a), which was
inhibited by the addition of CsA (Fig. 5b). IL-8 production
was modified by CBD (20 µM) alone (Fig. 5c) and the
addition of CsA increased IL-8 levels significantly (Fig. 5d).
MMP-3 levels were reduced by 20 µM CBD (Fig. 5e). The
cytokine-reducing effects of CBD might be related to the
reduction in viable cells, since we detected a reduction in
cell number at 10 µM and 20 µM CBD which was inhib-
ited by the addition of CsA (Fig. 5g, h).
Discussion
In this study, we demonstrated that CBD decreases cell
viability, proliferation, and cytokine production but
increases intracellular calcium and PoPo3 levels of RASF
and all effects were enhanced by TNF pre-stimulation.
These effects were mediated by TRPA1 and by the
assembly of the mPTP under pro-inflammatory condi-
tions, whereas under unstimulated conditions, TRPA1
was not involved.
We demonstrated that CBD reduces cell viability, but
RealTime-Glo assays were conducted in serum-free
medium without carrier protein. Therefore, we assessed
whether CBD influences RASF proliferation in medium
containing FCS, since in vivo, CBD is bound to serum
albumin, which lowers its free concentration available for
receptor binding
24,41
. We confirmed that the anti-
proliferative effect of CBD is dependent on FCS content.
Consequently, for in vivo applications, CBD needs to be
administered in high concentrations to elicit beneficial
effects as shown in the treatment of Dravet syndrome
13
.
In order to identify the cellular targets for CBD, we used
the TRPA1 antagonist A967079 and the pan-TRP
antagonist RR to inhibit the effects of CBD on cell via-
bility as CBD has already been identified as ligand for
TRPA1
23
. Moreover, we found that CsA reversed the
detrimental effects of CBD on cell viability, which con-
firms results from Olivas-Aguirre et al.
25
that showed
mitochondrial calcium overload correlates with assembly
of the mPTP by CBD. It has been demonstrated that CBD
influences calcium homeostasis
25
and we also found CBD
Fig. 5 IL-6, IL-8, MMP-3 production, and cell number after 72 h
incubation with CBD. RASF were incubated for 72 h with TNF. After
wash-off, RASF were challenged with antagonists for 30 min followed
by CBD addition for 72 h. ANOVA was used for all comparisons vs
control w/o CBD. a,c,e,gIL-6, IL-8, MMP-3 production, and cell
number after 72 h challenge with CBD. b,d,f,hIL-6, IL-8, MMP-3
production, and cell number after 72 h challenge with CBD (10 µM
and 20 µM) with concomitant addition of the TRPA1 antagonist
A967079 and the mPTP inhibitor CsA. Significant differences between
CBD in different concentrations are depicted as *p< 0.05, **p< 0.01,
and ***p< 0.001, and CBD versus CBD/antagonist treatment are
depicted as #p< 0.05, ##p< 0.01, and ###p< 0.001. ANOVA with
Bonferroni post-hoc test was used for all comparisons. The dotted line
in g,hrepresents the control value which was set to 100%. nis the
number of replicates out of four different patient samples. The error
bars represent the standard error of mean.
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to elevate intracellular calcium in RASF. This confirms
own previous results demonstrating calcium mobilization
in response to TRPA1 ligation
22
. Moreover, we showed
that TNF up-regulates TRPA1 protein in RASF, which
translates into increased sensitivity to TRPA1 ligands
22
.
CBD also increased calcium levels without extracellular
calcium by using PBS instead of HBSS, suggesting
mobilization form intracellular stores. In fact, in dorsal
root ganglia neurons it has been shown that TRPA1 is
located in lysosomes, where its activation fosters neuro-
transmitter release
42
. Although we do not provide direct
evidence regarding the localization of TRPA1, the use of
the cell-impermeable pan-TRP inhibitor RR
43
, which was
only able to slightly attenuate the effects of CBD suggests
an intracellular target protein. In line with this, the lipo-
philic antagonist A967079 decreased calcium mobiliza-
tion and PoPo3 uptake after CBD challenge. Moreover, we
used Thapsigargin to deplete ER calcium stores and GPN
to disrupt lysosomes and both compounds reduced the
elevation of intracellular calcium after CBD exposure.
This shows that ER calcium stores are involved and it has
been shown that even if calcium originates from lyso-
somes, the signal is amplified by depletion of ER stores
44
.
This is important, because although GPN has been
reported to mobilize lysosomal calcium, a recent study
claimed that GPN increases calcium through an ER-
dependent mechanism
45
. CBD is also an agonist at
TRPV1/2 ion channels
7,23,46,47
, but neither CPZ nor RR
inhibited the effects of CBD, ruling out these receptors as
target molecules. CPZ is also an agonist at TRPA1
48
, and
we did detect a small increase in basal intracellular cal-
cium in response to this ligand alone. Accordingly, CPZ
slightly elevated the calcium response of RASF to CBD
suggesting a sensitizing effect on TRPA1. TRPA1 inhibi-
tion with A967079 reduced calcium mobilization and
PoPo3 uptake in TNF pre-stimulated but not naïve RASF,
where we found the opposite, suggesting that CBD exerts
additional effects via different cellular targets besides
TRPA1. This demonstrates that TRPA1 contributes to the
rise in intracellular calcium only in TNF pre-stimulated
RASF, in which TRPA1 is upregulated. Besides binding to
TRPs, it has been demonstrated that CBD ligates several
proteins
11,49
with mitochondrial targets being the most
prominent
8–10,25,30
. In mitochondria, CBD targets
VDAC1, NCLX, MCU, and controls assembly of the
mPTP
8–10,25
. Although protective effects of CBD against
mitochondrial toxins have been shown
10,50
, the majority
of studies demonstrated that CBD induces cell death by
disturbing calcium homeostasis
8,9,25
. This confirms our
results, since CBD augmented calcium levels with a
concomitant increase in cell death. Excess cytosolic cal-
cium is taken up by mitochondria which are depolarized
in this process
51
. If mitochondrial calcium levels exceed a
certain threshold, mPTP is assembled leading to cell
death
52
. In fact, we demonstrated that only CsA prevented
cell death, suggesting that mitochondrial calcium over-
load occurs in CBD-stimulated RASF. In line with this,
CsA reduced intracellular calcium, which is another
indicator that mitochondria provide a significant con-
tribution to the increase in calcium by CBD. Calcium is
increased by mPTP formation as mitochondria are per-
meabilized releasing stored calcium into the cytosol
53
.
Thus, inhibiting mPTP formation by CsA decreases cal-
cium leakage from mitochondria and subsequent cell
death. In addition, we found that the NCLX inhibitor CGP
reduced cytosolic calcium levels. CGP blocks calcium
transport from the mitochondrial matrix into the inter-
membrane space, thus increasing mitochondrial and
reducing cytosolic calcium levels
54
. We also used the
reverse mode inhibitor of the NCLX, KB-R7943, but
results with this inhibitor are difficult to interpret due to
its interaction with the MCU and NCLX. In the latter it
can act as forward or reverse mode antagonist dependent
on cell type, NCLX isoform, and concentration
55,56
. Using
the specific MCU inhibitor DS16570511 we found
increased cytosolic calcium levels in TNF pre-stimulated
but not in unstimulated RASF. This might be explained by
TRPA1, which only contributed to calcium level altera-
tions in TNF pre-treated RASF. For the inhibition of
VDAC1 we used DIDS which increased intracellular cal-
cium in unstimulated, but decreased calcium in TNF pre-
treated RASF. This might depend on the initial calcium
signal generated by CBD, because DIDS can permeabilize
the inner mitochondrial membrane depending on calcium
concentration leading to formation of mPTP
57
. Besides
calcium, PoPo3 uptake served as readout for changes in
cell viability as it is supposed to enter cells with a com-
promised plasma membrane only. However, several stu-
dies showed that the uptake of PoPo3 related compounds
also occurs via specific receptors/ion channels
58,59
.In
addition, it has been demonstrated that the family of
organic cation transporters (OCT) mediates the uptake of
many charged but also electroneutral compounds into the
cell
39
. Therefore, it is quite possible that PoPo3 is also
taken up by OCT and indeed we show that decynium-22,
which inhibits all OCT isoforms
39
strongly reduced
PoPo3 uptake and it also blunted the increase of intra-
cellular calcium, which might be due to the electrogenic
properties of D22
40
. Another possibility is that OCT
mediates the uptake of CBD and D22 would limit the
access of CBD to intracellular compartments. D22 did not
influence basal uptake of PoPo3, but reduced the CBD-
induced uptake and this might be related to changes in
intracellular calcium since the activity of OCT is regulated
by calcium-dependent proteins
39
. DIDS completely
blocked PoPo3 uptake but these results are difficult to
interpret since DIDS does not inhibit OCT but membrane
anion channels, which should not mediate the uptake of
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the cationic dye PoPo3. It might be that the negatively
charged DIDS binds PoPo3 directly, thereby inhibiting
binding to DNA and the increase in fluorescence. PoPo3
might be suitable as a surrogate marker for the uptake of
chemical compounds/drugs, which is enhanced by CBD.
Since RASF produce high amounts of IL-6, IL-8, and
MMP-3
60
, we also investigated the impact of CBD on
production of these mediators. CBD dose-dependently
reduced IL-6, IL-8, and MMP-3 with concomitant
reduction in cell viability. CsA was able to rescue RASF
from cell death and increased Il-6 and IL-8 production
confirming that cell death is the influencing factor on
cytokine production.
From our data, we propose a mechanism of how CBD
influences RASF function and induces cell death under pro-
inflammatory conditions (Fig. 6). TNF sensitizes RASF to
the action of CBD by up-regulating TRPA1
22
.CBD
increases intracellular calcium by activating TRPA1 but it
also binds several mitochondrial targets like VDAC1, MCU,
and NCLX, which on their part influence cytosolic calcium.
Eventually, mitochondrial calcium overload occurs and
mPTP is assembled leading to cell death.
CBD has been used in animal model of RA, demon-
strating anti-inflammatory and analgesic effects but the
mechanism of action has not been identified
17,19
. Here, we
demonstrate that CBD reduces cell viability preferentially
in TNF-activated RASF via TRPA1 and mitochondrial
targets. CBD might decrease chronic inflammation since
RA is characterized by a hypoxic environment in the joint
with concomitant mitochondrial dysfunction
61
. In this
setting, immune cells and RASF might be specifically
vulnerable to a “second hit”induced by CBD, leading to
deletion of pro-inflammatory immune cells and fibro-
blasts thereby resolving inflammation. In addition, CBD
might also synergize with anti-rheumatic drugs like
methotrexate or JAK inhibitors, since it has been reported
in tumor cell lines that CBD works in synergy with e.g. the
chemotherapeutic drug doxorubicin
62
. Furthermore, CBD
also targets TRPV2, which not only increases the uptake
of cytotoxic chemotherapeutic agents but also reduces
RASF invasion and matrix metalloproteinase produc-
tion
47,63
. In conclusion, CBD might be beneficial as an
adjuvant treatment in rheumatoid arthritis that might
support the action of currently used disease-modifying
anti-rheumatic drugs.
Materials and methods
Biochemicals
Patients
In total, 40 patients with long-standing RA fulfilling the
American College of Rheumatology revised criteria for RA
(24) were included in this study. The RA group comprised
of 32 females and 8 males with a mean age of 67.8 years
±10.5 years and 66.9 years ±8.2 years, respectively. C-
reactive protein was 47.9 mg/dl ± 186.3 mg/dL for females
and 28.7 mg/dL ± 43.2 mg/dL for males and rheumatoid
factor was 184.4 iU/mL ± 280.4 iU/mL for females and
31.8 iU/mL ± 37.6 iU/mL for males. IN all, 11 out of 40
patients received glucocorticoids, 7 out of 40 methotrex-
ate, 3 out of 40 biologicals, and 1 out of 40 a JAK inhibitor.
All patients underwent elective knee joint replacement
surgery, and they were informed about the purpose of the
study and gave written consent. The study was approved
by the Ethics Committees of the University of Düsseldorf
(approval number 2018-87-KFogU) and Regensburg
(approval number 15-1 01-021). We confirm that all
experiments were performed in accordance with relevant
guidelines and regulations (Table 1).
Synovial fibroblast and tissue preparation
Samples from RA synovial tissue were isolated and
prepared as described previously
22
(for details see also
Supplementary methods).
Proliferation of RASF
Proliferation was assessed by the cell titer blue viability
assay (Promega, Madison, USA, # G8080) according to
manufacturer’s instructions.
Intracellular calcium and PoPo3 uptake
In black 96-well plates, RASF were incubated with 4 µM of
calcium dye Cal-520 (ab171868, abcam, Cambridge, UK) in
Hanks buffered salt solution (1 mM Ca
2+
;HBSS,sigma,#
Fig. 6 Proposed hypothetical mechanism of CBD-induced cell
death. TNF increases TRPA1 protein, which is located in intracellular
compartments. CBD activates TRPA1 and Ca
2+
is released into the
cytosol. Elevations in cytosolic Ca
2+
are reduced through uptake into
mitochondria. In addition, increased cytosolic Ca
2+
might enhance
the activity of organic cation transporters (OCT) which might mediate
the uptake of the fluorescent dye PoPo3. Additionally, OCT might
mediate the uptake of CBD itself. By binding to VDAC1, CBD increases
Ca
2+
flux through the outer mitochondrial membrane. Ca
2+
is then
taken up into the matrix by the mitochondrial Ca
2+
uniporter (MCU)
and, if mitochondria are depolarized, by the Na
+
/Ca
2+
exchanger
(NCLX), which operates in reverse mode under these conditions. Ca
2+
overload occurs, the mitochondrial permeability transition pore
(mPTP) assembles and cell death occurs.
Lowin et al. Cell Death and Disease (2020) 11:714 Page 8 of 11
Official journal of the Cell Death Differentiation Association

55037 C) or PBS (no Ca
2+
) with 0.02% Pluoronic F127
(Thermo fisher scientific, Waltham, USA, # P6866) for
60 min at 37 °C followed by 30 min at room temperature.
After washing, HBSS or PBS containing 1 µM PoPo3 iodide
(Thermo fisher scientific, # P3584) and respective antago-
nists/ligands/inhibitors were added for 30 min at room
temperature. After that, CBD was added and the intracellular
Ca
2+
concentration as well as PoPo3 uptake were evaluated
with a TECAN multimode reader over 90 min.
Flow cytometry
RASF were trypsinized, washed and fixed for 20 min
with 3.7% formaldehyde (F8775, Sigma Aldrich). Cells
were permeabilized with 0.1% Triton X-100 (X100,
Sigma) in PBS for 10 min. Then, 0.2 µg/50 µl primary
antibody (Proteintech, 19124-1-AP) was added for 2 h.
The secondary antibody (Abcam, goat anti-rabbit IgG
H&L (Alexa Fluor®488), ab150077) was incubated for
1 h. Cells were analyzed using a MACS Quant 9 analyzer
(Miltenyi Biotec, Bergisch Gladbach, Germany).
RealTime-Glo cell viability assay
Cell viability was assessed according to manufacturer’s
instructions (Promega, # G9711).
Statistical analysis
Statistical analysis was performed with SPSS 25 (IBM,
Armonk, USA). The statistic tests used are given in the
figure legends. Normal distribution was determined using
the Shapiro–Wilk test, equal variance was determined by
Levene’s test. In the case of equal variance, the Bonferroni
post-hoc test was used, otherwise the Dunnet’s post-hoc
test was employed. When data are presented as box plots,
the boxes represent the 25th to 75th percentiles, the lines
within the boxes represent the median, and the lines
outside the boxes represent the 10th and 90th percentiles.
When data are presented as line plots, the line represents
the mean. When data are presented as bar charts, the top
of the bar represents the mean and error bars depict the
standard error of the mean (sem). The level of significance
was p< 0.05.
Acknowledgements
We thank Birgit Opgenoorth for excellent technical assistance. This work was
supported by an unlimited grant of the Hiller Foundation. Open access
funding provided by Projekt DEAL.
Data availability
The datasets used and/or analysed during the current study are available from
the corresponding author on reasonable request.
Conflict of interest
The authors declare that they have no conflict of interest.
Ethics approval
This study was approved by the local ethics committees of Düsseldorf (2018-
87-KFogU) and Regensburg (15-1 01-021).
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Supplementary Information accompanies this paper at (https://doi.org/
10.1038/s41419-020-02892-1).
Received: 26 February 2020 Revised: 6 August 2020 Accepted: 6 August
2020
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CGP37157 1114 Tocris DMSO 1 µM, 10 µM
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