Hydroxychloroquine Is A Novel Therapeutic Approach For Rosacea

Hydroxychloroquine is a novel therapeutic
approach for rosacea

Author links open overlay panelJiLiabcdeXinYuanaYanTangadBenWangadZhiliDengabcdYingxueHuangadFangfenLiuadZhixiangZhaoadYiyaZhangabd
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Highlights
•
We revealed the potential molecular mechanism by which HCQ improved
rosacea in vivo and vitro.
•
The effects of HCQ treatment for rosacea patients were investigated.
•
HCQ is an effective treatment for rosacea.
Abstract
Rosacea is a chronic inflammatory disease in face. Hydroxychloroquine (HCQ), an
anti-malaria drug, was reported to have anti-inflammation activities. However, the
role of HCQ on rosacea remains unclear. In this study, we revealed the potential
molecular mechanism by which HCQ improved rosacea in rosacea-like mice and mast
cells (MCs). Moreover, the effects of HCQ treatment for rosacea patients were
investigated. In this study, we found HCQ ameliorated the rosacea-like phenotype and
MCs infiltration. The elevated pro-inflammatory factors and mast cell protease were
significantly inhibited by HCQ treatment in rosacea-like mice. In vitro, HCQ
suppresses LL37-induced MCs activation in vitro, including the release of
inflammatory factors, chemotaxis, degranulation and calcium influx. Moreover, HCQ
attenuated LL37-mediated MCs activation partly via inhibiting KCa3.1-mediated
calcium signaling. Thus, these evidences suggest HCQ ameliorated rosacea-like
dermatitis may be by regulating immune response of MCs. Finally, the 8-week HCQ
treatment exerted satisfactory therapeutic effects on erythema and inflammatory
lesions of rosacea patients, indicating that it is a promising drug for rosacea in clinical
treatment.
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Keywords
Rosacea
Hydroxychloroquine
Mast cells
Inflammation
Calcium signaling
Clinical treatment
Abbreviations
HCQ
hydroxychloroquine
MCs
mast cells
TLR2
toll-like receptor 2
Camp
Cathelicidin antimicrobial peptide
CEA
Clinician’s Erythema Assessment
IGA
Investigator’s Global Assessment
1. Introduction
Rosacea is a chronic inflammatory disease in face. Facial erythema, telangiectasia,
papules/pustules and recurrent flushing are major phenotype of the disease [1]. The
prevalence of rosacea is from 2% to 22% among different population [2], [3], [4]. The
etiology and pathogenesis of rosacea is not fully understood, innate immunity,
neuroimmunity and vascular dysfunction have been found involving [5]. Although it
is not a lethal disease, due to discosmetic dermatosis at the central face, which
significantly diminishing quality of life of rosacea patients [6].
Although the pathophysiology of rosacea is complex and remains poorly understood,
an accumulating number of studies consider the dysregulation of the immune system
to be a key factor in the pathogenesis of rosacea [7], [8], [9]. MCs are important effect
cells in host defense via inducing the expression of pro-inflammatory factors,
including IL-1β, TNF-α, IL-6, IL-17A, CCL2, and CXCL2 [10], [11], [12], [13].
Recent studies have reported the infiltration of MCs in rosacea skin lesions, and both
depleting and inhibiting the activation of MCs was found to substantially suppress
LL37-induced rosacea-like inflammation, indicating that MCs play a key role in
rosacea pathogenesis [12], [14], [15]. Moreover, a randomized clinical trial involving
10 rosacea patients showed that MCs were an effective therapeutic target for
rosacea [12].
Hydroxychloroquine (HCQ) was FDA-approved for lupus in 1955, is currently
frequently used to treat patients with systemic autoimmune diseases [16] and
considered safe during pregnancy [17], [18]. In these diseases, HCQ acts as an
immune modulator via suppressing immune cell function (e.g., macrophages, DCs, B
cells, and T cells) and reducing the secretion of pro-inflammation
cytokines [19], [20], [21], [22]. Moreover, HCQ is thought to function via the
inhibition of MC infiltration [23], suppression of angiogenesis [20], [24], [25], and
protection of cells from UVB exposure [26], which have been implicated in the
pathogenesis of rosacea. Therefore, these evidences suggest that HCQ may be a
potential therapeutic for the treatment of rosacea.
In the present study, we investigated the role and potential mechanism of HCQ on
rosacea treatment in vitro and in vivo. Our findings demonstrated that the therapeutic
mechanism of HCQ on rosacea to be mediated partly by reducing the MC-associated
inflammatory response. Moreover, the erythema and inflammatory of rosacea patients
were significantly attenuated by a 8-week HCQ treatment. These results indicate that
HCQ is a promising therapy for rosacea treatment.
2. Materials and methods

2.1. Animal experiments
BALB/c (7-week-old) mice were obtained from Slac Laboratory Animal Co. Ltd.
(Shanghai, China). LL37 was synthesized by Shenggong (Shanghai, China), identified
by HPLC and purity was more than 95%. For HCQ (Selleck Chemicals, California,
USA) administration, 40 mg/kg HCQ was administered daily by gavage for seven
consecutive days. To conduct a rosacea-like mouse model, mouse back was shaved
24 h before treatments, and 40 μl of LL37 (320 μM) was administered by means of
intra dermal injection (twice a day) at the last 2 days as previous described [8]. The
mouse lesions were photographed and erythema was evaluated as described in our
previous study [7]. All animal experiments were approved by the Animal Ethics
Committee of the Xiangya Hospital of Central South University.
2.2. Skin MC culture
Mouse skin MCs were collected and cultured as previously described [27]. Briefly,
the skin of the newborn mice was digested in 10% collagenase type IV, and 5%
isolation enzyme 2. The primary mouse skin cells were cultured in RPMI 1640
medium (Gibco, Thermo Fisher Scientific, USA) containing 500 mol/L β-
mercaptoethanol, 100 mmol/L non-essential amino acid, 100 mmol/L sodium
pyruvate, 100 IU/mL penicillin, 100 mg/mL streptomycin, 0.25 mg/mL amphotericin
B mixed solution, 20 mmol/L glutamine, 250 mmol/L Hepes, 100 mL/L fetal bovine
serum (Gibco, ThermoFisher Scientific, USA), 10 ng/mL IL-3 (PeproTech, Inc,
USA), and 10 ng/mL rmSCF (PeproTech, Inc, USA) in an incubator at 37 ℃ and 5%
CO2 without changing the medium. Two weeks later, MCs were consistently
generated and separated by Percoll (40% isotonic Percoll + 60% culture medium),
then centrifuged at 500g for 10 min at room temperature. MCs were confirmed by the
expression of FceRI and CD117 (c-Kit) and the purity was greater than 95%. For
HCQ treatment, 2 × 106 MCs were pre-treated with HCQ (10 μM) for 1 h, then
stimulated with LL37 (8 μM) for 6 h at 37 °C.
2.3. Histological analysis
For histological analysis, 4-mm mouse lesion specimens were fixed overnight in 4%
buffered formalin, embedded in paraffin, sectioned, then stained with hematoxylin-
eosin or toluidine blue. Finally, the sections were observed using an optical
microscope (Olympus, Japan).
2.4. Immunofluorescence
Frozen mouse lesion sections were fixed with 4% paraformaldehyde for 15 min and
washed with phosphate-buffered saline (PBS), blocked with 5% BSA (Shenggong,
Shanghai, China) which containing 0.3% TritonX-100 for 1 h at room temperature,
then treated with anti-Mast Cell Tryptase antibodies (1:200, Abcam, Cambridge,
United Kingdom) at 4 °C overnight. Alexa Fluor 488-labeled anti-goat IgG antibodies
were used as the secondary antibody for 1 h on the following day, then lesion sections
were washed and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for
5 min. A Zeiss Axio Scope A1 (Zeiss, Germany) was used to acquire the images.
2.5. Cell viability assay
Mast cells (2 × 103) were cultured in a 96-well plate and treated with various HCQ
concentrations (0 μm, 10 μm, 25 μm, 50 μm and 100 μm) for 24 h. After removing the
supernatant of each well, 10 μl CCK8 solution and 100 μl media were introduced.
After incubation for 1 h in 5% CO2 at 37 ℃ for 1 h, the cell viability was evaluated by
measuring the absorbance intensity of the cell suspension at 450 nm by a microplate
reader (PerkinElmer EnSight).
2.6. qPCR analysis
According to the instructions, 1 μg of RNA extracted from cell or tissue was reverse
transcribed into cDNA using PrimeScript RT reagent Kit (Takara, Shiga, Japan), and
RT-PCR was conducted with SYBR GREEN (Bio-Rad, California, USA) on a 7500
machine (Applied Bio systems, Life Technologies) 7. The primer sequences are listed
in Table 1.
2.7. MCs chemotaxis assay
8-µm pore size transwell chambers (Millipore, Billerica,MA) were used to measure
MCs migration. In a 24-well culture plates, 500 μl 1640 medium with 10% FBS,
LL37 (4 μM) or equal amount of PBS was added into each lower chamber. MCs
(2 × 103) with 200 μl 1640 medium without FBS co-incubated with 10 μM HCQ or
TRAM-34 (300 nM) were seeded into each upper chamber. Cells were cultured in an
incubator at 37℃ and 5% CO2. After 24 h, MCs adherent to the upper surface of the
membrane were scraped using a cotton bud, cells on lower surface were fixed with
4% paraformaldehyde for 15 min and stained with 0.5% toluidine blue for 30 min.
Cell numbers on the lower membrane were counted under an optical microscope
(OLYMPUS, Japan) in three random visual fields.
2.8. Degranulation experiment
Degranulation of MCs was measured by detecting the amount of β-hexosaminidase

released into the medium. MCs (1 × 105) were seeded with Tyrode’s buffer in 12-well
plates. The cells were pretreated with 10 µM HCQ or TRAM-34 (300 nM) for 60 min
an incubator at 37 °C and 5% CO2, and LL37 (8 μM) was used to stimulate the cells
for 1 h at 37 ℃. Then, the supernatant (50 µl) was transferred to a 96-well plate and
co-incubated with 50 µl of 2 mM PN-(GlcNAc) 2 for 2 h at 37 ℃. The reaction was
terminated by adding 200 μl of 0.4 M glycine (pH 10.7). The absorbance was
measured with a Multimode Plate Reader at 405 nm (PerkinElmer EnSight). The total
cellular β-hexosaminidase was detected by the equal number of MCs lysed with 1%
Triton X-100.
2.9. Intracellular calcium detection
MCs (1 × 106) were pre-incubated with 2 µM Fura-2 AM for 30 min at 37 °C then
washed 3 times with no-calcium Tyrode’s buffer and re-suspended in Tyrode’s buffer.
The cells were treated with 10 μM HCQ or TRAM-34 (300 nM) for 30 min and
200 μl (2 × 105 per wells) cells were transferred to a black 96-well plate. The
fluorescence (340 nm/380 nm) was detected for 30 s and LL37 (8 μM) was adding
immediately, then the fluorescence (340 nm/380 nm) was detected by 10 s intervals
by a Multimode Plate Reader (PerkinElmer EnSight) to 5 min.
2.10. Clinical evaluation for HCQ
We conducted a trial in 6 adult patients with moderate to severe rosacea (ETR,

erythematotelangiectatic rosacea and PPR, papulopustular rosacea) fora 8-weeks HCQ
treatment. The following patients were excluded from the study: patients who were
pregnant, lactating, or recently planning to become pregnant; patients who had a
history of moderate to severe liver, kidney, lung, or blood conditions, other systemic
diseases, or mental disorders; patients who were combined with nasal hypertrophy of
hypertrophic rosacea patients; patients with facial manifestations of other facial skin
diseases or other diseases; patients who had participated in any clinical trial during the
previous month, patients with an allergy to chloroquine; patients who had used
glucocorticoids, antibiotics, or Victoria A acid within the past three months; and
patients who had used glucocorticoid ointment, tacrolimus (pimecrolimus), laser and
other treatment within one month were excluded. The patients without ANA (Serum
antinuclear antibody) abnormal, systemic disease and potential retinopathy were
assigned to receive oral HCQ (200 mg twice daily) for 8 weeks. These participants
underwent follow visits: baseline/screening, and at weeks 4 and 8. Patients were
assessed by dermatologists for inflammatory lesions with Investigator’s Global
Assessment (IGA), on a 5point scale (0 = Clear: 0 papules/pustules; 1 = Near clear: 1–
2 papules/pustules; 2 = Mild: 3-10papules/pustules; 3 = Moderate:11–19
papules/pustules; 4 = Severe > 20papules/pustules),and for facial erythema with a
Clinician’s Erythema Assessment (CEA), on a 5 point scale(0 = None; 1 = Mild;
2 = Moderate; 3 = Significant; 4 = Severe) [28]. The patients who have an history of
stable rosacea with CEA ≥ 2 or/and IGA ≥ 3 were included in trial. At 8th week,
successful treatment of facial inflammatory lesions and erythema were defined as IGA
score 0 or 1 and CEA score 0 or 1, respectively. In the whole clinical trial, the patients
were not received other drug treatment except HCQ. Adverse events (AEs) were
recorded at each visit. The study was approved by the National drug clinical trial
institutions-Central South University Xiangya Hospital Ethics Committee under IRB
number ChiCTR-IPR-17012224. All participants provided written informed consent.
2.11 Statistical analyses
All data were presented as means ± SEM. The ANOVA with LSD post-test analyzed
with Spss 17.0 was used for compare differences between different groups. *,
P < 0.05;**, P < 0.01 and ***, p < 0.001 are considered significant.
3. Result
3.1. HCQ attenuated rosacea-like dermatitis in an LL37-induced mouse model
We investigated the therapeutic effects and pharmacological mechanisms of HCQ on

rosacea using an LL37-induced rosacea-like mouse model. As shown in Fig. 1a–c,
HCQ significantly attenuated LL37-induced rosacea-like erythema. The average
redness score and redness area were reduced by approximately 57% and 35%,
respectively. The histological analysis demonstrated that skin edema and
inflammatory infiltration in rosacea-like dermatitis were also ameliorated by HCQ
treatment (Fig. 1d and e). Furthermore, we investigated the effects of HCQ on the
expression of rosacea-related inflammatory factors. As shown in Fig. 1f, HCQ
substantially suppressed the LL37-induced expression of CAMP, TNFα, IL1β, IL-6,
and IL-17A in rosacea-like skin lesions. Thus, these results indicated that HCQ
ameliorated the symptoms of rosacea in an LL37-induced mouse model.
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Fig. 1. HCQ treatment ameliorated rosacea-like dermatitis in mice. (a) LL37 was
intradermally injected into the dorsal skin to induce a rosacea-like phenotype. HCQ was
pretreated by gavage (b) and (c) the severity of the rosacea-like phenotype was assessed
based on the redness score and area. (d) and (e) H&E staining for the histological analysis
of rosacea-like skin. Scale bar: 100 μm. (f) The expression levels of CAMP, TNFα, IL-
1β, IL-6, and IL-17A in mouse lesions was detected by qPCR analysis. Results are
representative of three independent experiments. n (CON and HCQ group) = 6, n (LL37
and LL37 + HCQ group) = 10, data represent the means ± SEM; * p < 0.05; **
p < 0.01; and *** p < 0.001.
3.2. HCQ suppressed LL37-induced MC infiltration and protease expression in

rosacea-like mice
An accumulating number of studies suggest that MCs play an important role in the
development of inflammatory dermatosis, including rosacea [29]. MC infiltration was
observed in the skin lesions of rosacea patients and mice [12], [30]. Moreover, LL37-
induced proteases released from MCs are crucial in the development of rosacea
inflammation [12]. Here, we found that HCQ suppressed LL37-induced MC
infiltration (Fig. 2a and b) and mast cell tryptase (Fig. 2c and d). In addition, we also
detected the experiment of mast cell protease in mouse lesions by qPCR. As shown
in Fig. 2e, LL37-induced CMA1, Tpsab1 (the gene for chymase and tryptase), and
MMP9 expression were inhibited by HCQ treatment in mouse skin lesions.
Collectively, our research suggested that HCQ attenuated rosacea-like inflammation
partially by reducing MC infiltration and protease release.
Fig. 2. HCQ treatment inhibited MC infiltration in rosacea-like dermatitis. (a) Toluidine
blue staining revealed the infiltration of MCs in rosacea-like mice. Scale bar: 100 μm (b)
MC tryptase (Tpsab1) expression in the skin visualized by immunofluorescence. Green
indicates Tpsab1; blue indicates DAPI; scale bar: 100 μm. (c) and (d) MC-related
protease expression in rosacea-like mice. (e) Rosacea-related proteases were detected by
qPCR. Results are representative of three independent experiments. n (CON and HCQ
group) = 6, n (LL37 and LL37 + HCQ group) = 10, data represent the means ± SEM. *
p < 0.05; ** p < 0.01; and *** p < 0.001.
3.3. HCQ suppresses LL37-induced MC activation in vitro
We found that the concentrations of 100 μM HCQ presented significantly negative

effect on viability of mast cells. (Fig. 3a) and we chose a drug concentration of 10 μm
in cell experiment. We next examined the effects of HCQ on the expression of
inflammatory factors in MCs. We found that HCQ significantly reduced LL37-
induced IL-1β, IL-17A, TNFα, CXCL2, CCL2, Tpsab1, and MMP9 expression in
MCs (Fig. 3b). Consistent with a previous report 23, HCQ did not substantially
suppress CMA1 expression in MCs (Fig. 3b). Thus, together these results indicated
that HCQ ameliorated the LL37-induced activation of MCs. Several studies have
shown that LL37 is an MC chemotactic factor that can stimulate MC
degranulation [31], which plays an important role in rosacea inflammation. In the
present study, we found that HCQ exerted the substantial suppression of LL37-
inducedMC chemotaxis (Fig. 3c and d) and degranulation (Fig. 3e).
Fig. 3. HCQ significantly inhibited LL37-mediated MC activation. (a) The effect of
different concentrations of HCQ on cell stability in CCK8 experiment. (b) Rosacea-
related inflammatory factors were detected by qPCR. (c) and (d) The chemotaxis ability
of MCs was detected using a Transwell assay. (e) β-hexosaminidase was detected by the
level of absorbance at 405 nm. Results are representative of three independent
experiments. n = 3, data represent the means ± SEM.* p < 0.05; ** p < 0.01;and ***
p < 0.001.
3.4. HCQ attenuates LL37-mediated MC activation via inhibiting KCa3.1-

mediated calcium signaling
Calcium influx plays a critical role on MC pro-inflammatory factor release,

degranulation, and chemotaxis [32], [33]. MrgprB2 (Mas-related Gene X2) and the
Ca2+ -activated K + channel (KCa3.1) play a key role in regulating the calcium influx
in MCs. Since HCQ exerted inhibitory effects on MCs, we speculated that HCQ could
inhibit MrgprB2/KCa3.1-mediated the calcium influx in MCs. As shown in Fig. 4a
and b, HCQ markedly suppressed LL37-induced calcium influx and reduced LL37-
induced KCa3.1 expression in MCs; however, LL-37-induced MrgprB2 was not
affected by HCQ treatment. For further confirmation, we used a KCa3.1 inhibitor
(TRAM-34, a derivative of clotrimazole and a highly specific blocker of Kca3. 1) for
subsequent experiments. Similar with HCQ, TRAM-34 also suppressed LL37-
promoted calcium influx (Fig. 4c) degranulation (Fig. 4d) and chemotaxis and (Fig. 4e
and f) in MCs. These results indicated that HCQ might suppress the activation of MCs
via decreasing KCa3.1-mediated calcium signaling.
Fig. 4. HCQ inhibited LL37-induced MC activation via suppressing Ca2+ signaling. (a)
The level of intracellular calcium in MCs in HCQ experiments. (b) The effects of HCQ
and LL37 on KCa3.1andMrgprB2 expression in MCs. (c) The level of intracellular
calcium in MCs in TRAM-34 experiments. (d) β-hexosaminidase was detected by the
level of absorbance at 405 nm. (e) and (f) The chemotaxis ability of MCs was detected
using a Transwell assay. Results are representative of three independent experiments.
n = 3, data represent the means ± SEM. *p < 0.05; **p < 0.01; and ***p < 0.001.
3.5. HCQ exerted therapy effects on facial inflammatory lesions and erythema in

patients
After 8 weeks of HCQ treatment, patients achieved phenotypic improvement of
rosacea (Fig. 5a). In addition, as shown in Fig. 5b and c, IGA and CEA score showed
a tendency to relief of rosacea. Inflammatory lesions and erythema success ratio were
67% and 83%, respectively. During 8 weeks of HCQ treatment and 4 weeks after
HCQ treatment, there were no obvious adverse reactions (including no discomfort of
eyes) for patients.

Fig. 5. The oral administration of 200 mg HCQ twice daily for 8 weeks exerted an
evident therapeutic effect in patients. (a) Photo of patients at 0 week and 8th week. (b)
The change of patients’ IGA score showed in stacked histogram at 0 week, 4th week and
8th week. (c) The change of patients’ CEA score showed in stacked histogram at 0 week,
4th week and 8th week. (d) The change of patients’ papules/pustules at 0 week, 4th week
and 8th week.
4. Discussion
To date, the efficacy and safety of treatment options for rosacea remain limited. HCQ
is a widely used classical anti-malarial and anti-rheumatic drug that is prescribed as a
therapy for various cutaneous diseases as it inhibits MCs [23],
angiogenesis [20], [24], [25], and protects skin from UVB [26]. These risk factors also
have been implicated in the pathogenesis of rosacea. However, the therapeutic
mechanism of HCQ for rosacea remains unknown. In the present study, we found that
HCQ attenuated the rosacea-like dermatitis partly by repressing MCs mediated
inflammation. Moreover, HCQ showed therapy effects on rosacea patient by 8 weeks
treatment, which providing a valuable option for further clinical treatment on rosacea.
MCs are important effector cells required for host defense via the production and
release of pro-inflammatory cytokines and chemokines [10].Recent studies have
shown that there are an increased number of MCs in the skin lesions of patients with
rosacea and inhibiting MC function attenuates rosacea-like skin
inflammation [12], [14], [15], indicating that MCs are a potential therapeutic target for
rosacea. Moreover, treatment with an MC stabilizer (i.e., cromolyn sodium) was
reported to decrease the levels of facial erythema in rosacea patients, and
onabotulinumtoxin [12] was reported to reduce rosacea-associated skin inflammation
by inhibiting MC degranulation [34]. In the present study, we found that HCQ
suppressed MC infiltration and protease expression in rosacea-like mice. Because MC
activation plays a key role in the inflammatory response and vascular permeability in
the skin [35], HCQ may reduce the erythema and inflammation associated with
rosacea partly by suppressing MC activation. Recent studies have revealed that the
therapeutic effects of HCQ on inflammatory disease are achieved by directly
regulating the function of immune cells [36], including MCs [23]. Consistent with
these studies, we found that HCQ effectively suppressed LL-37-mediated MC
activation. Both calcium (Ca2+) flux and Ca2+-dependent signaling play a crucial role in
various biological process, including the activation of MCs. The Ca 2+ influx in MCs
can be directly regulated by KCa3.1 [37], which has been reported to be a potential
therapeutic target for diseases involving MC activation [38]. In our research, we also
found HCQ may attenuate LL37-induced MC activation via suppressing KCa3.1-
mediated local Ca2+ influx, based on TRAM-34, a highly specific blocker of KCa3.1,
showed similar effects of HCQ on LL37-activiate mast cells. In addition, HCQ
seemed to inhibit the expression of KCa3.1, these findings could further support HCQ
attenuate LL37-induced MC activation may partly via KCa3.1. We consider HCQ
may bind to some crucial receptor of KCa3.1 and effect its expression, however more
in-depth researches are needed. With regard to our study, experimental results in vitro
indicated that HCQ may attenuate LL37-induced MC activation via suppressing
KCa3.1-mediated local Ca2+ influx, thereby reducing the augmented chemokine
production and recruitment of neutrophils and monocytes.
In this study, we found that 200 mg HCQ administered twice daily was effective for
rosacea patients with inflammatory lesions and erythema. Moreover, after 8-weeks
HCQ treatment, there were no obvious adverse reactions for patients. Although
Hydroxychloroquine has potential ocular toxicity in long-term use (for more than one
year) and no cases of retinopathy have been found in this study. Hydroxychloroquine
is used for the treatment of rosacea for only two months in this work, patients prevent
the recurrence of rosacea through moisturizing and sun protection after HCQ
treatment. These data indicated that HCQ may be a good candidate for patients with
rosacea, however more patients are needed to confirm these results and safety.
In summary, these findings suggested that the therapeutic mechanism by which HCQ
inhibited the infiltration and activation of MCs was via KCa3.1-mediated
Ca2+ signaling, and it is a promising drug for rosacea in clinical treatment. However,
large scale clinical trial is needed to further verify the safety and effectiveness of HCQ
in the treatment of rosacea.
Author contribution
Yiya Zhang and Ji Li designed research; Xin Yuan, Zhixiang Zhao, Xingxue Huang
and Ben Wang performed research; Yan Tang, Zhixiang Zhao, and Fangfen Liu
contributed new reagents or analytic tools; YiyaZhang and Xin Yuan analyzed data; Ji
Li, Yiya Zhang and Xin Yuan wrote the paper.
Acknowledgments
This work was supported by the National Natural Science Foundation of
China (No. 81703149, No. 81602784, No. 81502709).
Declaration of Competing Interest

The authors declare no conflicts of interest.
Appendix A. Supplementary material

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Supplementary data 1.
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Hydroxychloroquine Is A Novel Therapeutic Approach For Rosacea

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2. Materials and methods

2.5. Cell viability assay

2.7. MCs chemotaxis assay

Degranulation of MCs was measured by detecting the amount of β-hexosaminidase

2.9. Intracellular calcium detection

2.10. Clinical evaluation for HCQ

We conducted a trial in 6 adult patients with moderate to severe rosacea (ETR,

2.11 Statistical analyses

We investigated the therapeutic effects and pharmacological mechanisms of HCQ on

3.2. HCQ suppressed LL37-induced MC infiltration and protease expression in

3.3. HCQ suppresses LL37-induced MC activation in vitro

We found that the concentrations of 100 μM HCQ presented significantly negative

3.4. HCQ attenuates LL37-mediated MC activation via inhibiting KCa3.1-

Calcium influx plays a critical role on MC pro-inflammatory factor release,

3.5. HCQ exerted therapy effects on facial inflammatory lesions and erythema in

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Declaration of Competing Interest

Appendix A. Supplementary material

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