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Journal Pre-proof

PAR2–β-arrestin 2–ERK axis mediates

Malassezia globosa

-induced IL-17 response

by disrupting ZO-1 in keratinocytes

Jinfeng Tang, Fanggu Li, Jiaqing Hong, Xueying Li, Ying Zhou, Jie Liu, Yiming Fan

PII:

S2589-0042(26)01021-7

DOI:

https://doi.org/10.1016/j.isci.2026.115646

Reference:

ISCI 115646

To appear in:

iScience

Received Date: 2 September 2025
Revised Date: 8 September 2025
Accepted Date: 6 April 2026

Please cite this article as: Tang, J., Li, F., Hong, J., Li, X., Zhou, Y., Liu, J., Fan, Y., PAR2–β-arrestin 2–

ERK axis mediates

Malassezia globosa

-induced IL-17 response by disrupting ZO-1 in keratinocytes,

iScience

(2026), doi:

https://doi.org/10.1016/j.isci.2026.115646

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PAR2

–β-arrestin

–ERK

axis

mediates

Malassezia globosa-induced IL-17 response by
disrupting ZO-1 in keratinocytes

Jinfeng Tang,

1,2,5

Fanggu Li,

2,5

Jiaqing Hong,

Xueying Li,

Ying Zhou,

Jie Liu,

and Yiming

Fan

2,4,6,

Clinical Research Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang, 524001, China

Department of Dermatology, Affiliated Hospital of Guangdong Medical University, Zhanjiang, 524001, China

Experimental Animal Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang, 524001, China

Dermatology, Plastic and Cosmetic Surgery Center, the First Dongguan Affiliated Hospital of Guangdong Medical

University, Dongguan, 523106, China

These authors contributed equally

Lead contact

*Correspondence: ymfan1963@163.com

SUMMARY

Malassezia

, a lipid-dependent commensal yeast of human skin, also functions as an opportunistic

pathogen contributed to the pathogenesis of various skin and systemic diseases. Although the

association between protease-activated receptor 2 (PAR2) and fungal infections has been partially

characterized, its specific role in

Malassezia

-related dermatoses remains poorly elucidated. In this study,

we demonstrated that PAR2 interacted with zonula occludens-1 (ZO-

1) and β-arrestin 2 to cooperatively

regulate

M. globosa

-infected keratinocytes by Co-IP, PLA, and MbYTH. Utilizing

Malassezia

folliculitis

patient samples, cellular infection models, and murine infection models, we demonstrated that

M. globosa

exploited the PAR2

–β-arrestin 2–ERK signaling axis by disrupting ZO-1, consequently amplifying IL-17-

mediated inflammatory responses. Notably, pharmacological inhibition or genetic ablation of PAR2

attenuated the

Malassezia

-induced IL-17 inflammation. Collectively, our results clarify PAR2 as a

potential therapeutic target for

Malassezia

-infected diseases, while providing new insights into the

pathogenic mechanisms of commensal fungi.

KEYWORDS

Protease-activated receptor 2,

Malassezia

globosa

, Tight junction, Interleukin-17, Inflammation

INTRODUCTION

The genus

Malassezia

currently comprises 18 recognized species and is the most abundant symbiotic

fungus on the skin of humans and homeothermic animals.

1,2

As both commensals and opportunistic

pathogens, they can cause several skin conditions including

Malassezia

folliculitis (MF), pityriasis

versicolor and seborrheic dermatitis, and aggravate atopic dermatitis, psoriasis, and even systemic

diseases (such as respiratory, neurological, gastrointestinal, and genital infections).

3-5

Among these

species,

M. globosa

is the main pathogen causing MF, pityriasis versicolor, and psoriasis.

6-10

Nevertheless, the precise pathogenic mechanisms underlying

M. globosa

-mediated diseases remain

poorly understood.

Malassezia

is a lipid-dependent dimorphic yeast that lacks lipid metabolism genes, but

it modifies the cutaneous microenvironment by secreting various hydrolytic enzymes such as protease,

lipase, phospholipase, and sphingomyelinase, which disrupt the epidermal barrier and facilitate microbial

colonization.

These fungi primarily colonize the stratum corneum and hair follicle infundibula.

Keratinocytes and immunocytes recognize

Malassezia

through pattern recognition receptors (PRRs),

subsequently initiating inflammatory signaling pathways.

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Keratinocytes are major epidermal components that play a crucial role in maintaining the intact skin

barrier and harmonious immune responses.

13-15

As active innate immunocytes, keratinocytes express

multiple PRRs that activate the microbial and host danger molecules and induce the release of

inflammatory cytokines, chemokines, and antimicrobial peptides, thereby initiating various immune

responses.

Malassezia

infection involves Th1, Th2, and Th17 immune responses.

1,17

Previous studies

found that

M. globosa

was the main

Malassezia

species in MF and promoted Th1 response of HaCaT

cells through NLRP3 inflammasome.

9,17

Emerging evidence suggests that

Malassezia

can trigger an IL-

17-dependent inflammatory cascade in keratinocytes,

contributing to the pathogenesis of some skin

diseases including contact dermatitis, chronic pruritus, dust mites-induced atopic dermatitis, and

Staphylococcus aureus

infection.

19-23

Malassezia

can aggravate skin inflammation through Th17-biased

immune response.

10,19

Keratinocytes participate in this process via IL-36/MYD88 axis, which drives IL-17-

dependent inflammation induced by

M. globosa

In addition,

M. globosa

promotes IL-23 secretion of

keratinocytes, which further amplifies pathogenic Th17 differentiation through TLR2/MYD88/NF-

κB

pathway.

Although cumulative data confirm the association between

Malassezia

infection and IL-17

signaling in keratinocytes,

the exact regulatory mechanism on this inflammatory response remains to be

clarified.

Malassezia

-evoked Th17 immune response has a dual effect on the host, both antifungal and

proinflammatory, possibly hinging on its symbiosis and pathogenicity.

25,26

In mouse model of skin

infection,

Malassezia

prevented fungal overgrowth through IL-23/IL-17 axis in intact skin but aggravated

IL-23/IL-17 axis-dependent skin inflammation in damaged skin.

In atopic dermatitis and psoriasis,

Malassezia

can

promote keratinocyte proliferation and exacerbate skin inflammation via triggering Th17

immune response.

Furthermore, the fungal infection (especially MF) risk increased following

administration of IL-17 inhibitors, implying that IL-17 is crucial for antifungal immunity.

Protease-activated receptor 2 (PAR2) belongs to the rhodopsin G protein-coupled receptors (GPCR)

receptor and is widely expressed in epithelial/endothelial cells, platelets, smooth muscle cells, nerve cells,

immunocytes.

29-32

PAR2 is primarily activated by serine proteases and plays a significant role in the

pathogenesis of coagulation, cardiovascular diseases, cancer, inflammatory skin diseases.

During

PAR2 activation, its N-terminal is firstly cleaved by proteases to expose a new N-terminal sequence TL,

followed by molecular docking with transmembrane receptors, resulting in intracellular signal

transduction.

PAR2 can act as a non-classical PRR, exerting immunoregulatory role in various fungal

infections.

34-36

Aspergillus fumigatus

and

Alternaria

can promote IL-6, TNF-

α and GM-CSF secretion in

airway epithelium via PAR2, inducing airway epithelial inflammation and allergic asthma.

34,36

Alternaria

serine protease can not only rapidly release IL-

33 by activating PAR2, but also activate PAR2/β-arrestins

pathway to induce Th2 immune response, thereby aggravating airway inflammatory response.

Candida

albicans

Sap6 activated PAR2 through RGD domain to initiate release of IL-

1β and IL-8 in human oral

epithelial cells;

Paracoccidioides brasiliensis

induced secretion of IL-6 and IL-8 in lung epithelial cells

A549 by activating PAR2.

At present, the role of PAR2 in fungal infections mainly focuses on Th1 and

Th2 immune responses, with little attention to Th17 immune responses. It is unknown whether PAR2 is

involved in the pathogenicity of

Malassezia

infection.

In this study, we systematically investigated the effect of PAR2 on IL-17-driven inflammatory response in

Malassezia

-infected keratinocytes and mice.

RESULTS

Expression of PAR2, IL-17 and tight junction (TJ) proteins in Malassezia folliculitis

To elucidate the underlying mechanism of MF, we performed liquid chromatography-tandem mass

spectrometry (LC-MS/MS) analysis on three paired MF lesions and normal skin samples, followed by

comprehensive bioinformatics analysis to identify differentially expressed proteins (DEPs). Proteomic

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profiling revealed significant enrichment of DEPs in several pathways, including complement and

coagulation cascades, cell adhesion, extracellular matrix (ECM) receptor interactions, cytokines signaling,

and viral infection responses (Figure S2A-F). The most prominent DEPs were PAR2, IL-17, and key TJ

proteins (ZO-1, Occludin, Claudin-1), which were confirmed by immunohistochemistry. Compared with

normal skin (CTRL), PAR2, IL-17, and Occludin expression was increased, while ZO-1 and Claudin-1

expression was decreased in six MF lesions (Figure 1A, B). Therefore, these results suggest that PAR2,

IL-17, and TJ dysregulation may contribute to the pathogenesis of MF. Furthermore, periodic acid

–Schiff

(PAS) staining revealed that skin-colonizing spores were more abundant in MF than in normal skin

(Figure 1C, D).

Impact of M. globosa infection on PAR2 activation, IL-17 production and TJ integrity in HaCaT

cells

100

M. globosa

, the predominant pathogent in MF,

elicits diverse proinflammatory responses in

101

keratinocytes.

Emerging evidence suggests that keratinocytes orchestrate

Malassezia

-induced, IL-17-

102

mediated cutaneous inflammation via IL-36 and IL-23 signaling.

18,24

To delineate the functional

103

consequences of

M. globosa

infection on keratinocyte, we first evaluated cellular viability under varying

104

multiplicities of infection (MOI). CCK-8 assays revealed a significant dose-dependent reduction in HaCaT

105

cell viability at 50-200 MOI, whereas 0-30 MOI exhibited no detectable cytotoxicity (Figure 2A).

106

Consequently, 30 MOI was chosen for subsequent experiments to maximize infection efficiency and

107

preserve cellular homeostasis.

108

PAR2 has been implicated in the regulation of IL-17 across multiple infections and inflammatory contexts,

109

including

Helicobacter pylori

and

Porphyromonas gingivalis

infections, as well as chronic pain

110

disorders.

40-42

We therefore postulated that

M. globosa

might

similarly engage

PAR2-dependent pathways

111

in keratinocytes. Notably, PAR2 is known to modulate TJ dynamics in keratinocytes and

112

pulmonary/intestinal

epithelial cells through β-arrestin-mediated ERK1/2 phosphorylation, influencing both

113

barrier integrity and inflammatory responses.

43-46

To investigate this mechanism, we performed temporal

114

profiling of PAR2, β-arrestin 2, phospho-ERK (p-ERK), IL-17, and TJ proteins (ZO-1, Occludin, Claudin-1)

115

M. globosa-

infected HaCaT cells. Western blotting and qRT-PCR assays revealed significant temporal

116

modulation of these markers at 0.5, 4, and 24 hours post-infection (Figure 2B-F).

117

Furthermore, ELISA revealed a significant increase in IL-17 secretion 12 hours post-infection (Figure 2G),

118

indicating a time-dependent induction of IL-17 by

M. globosa

at 30 MOI. The peak IL-17 response

119

abserved at 24 hours post-infection paralleled our previous findings on

M. globosa

-driven Th1-polarized

120

immunity,

further hightlighting

the yeast’s ability to skew keratinocyte-mediated inflammatory networks.

121

Collectively, these data demonstrate that

M. globosa

infection may potentiate PAR2 signaling,

122

compromise TJ integrity, and enhance IL-17 release in keratinocytes, thereby fostering a proinflammatory

123

microenvironment conductive to MF pathogenesis.

124

Ultrastructural characterization of HaCaT cells and M. globosa interaction during infection

125

M. globosa

typically maintains skin homeostasis through complex microenvironmental interactions,

but

126

can transition into a pathogenic state under cutaneous dysbiosis, leading to

Malassezia

-associated

127

dermatoses.

To characterize the cytopathological effects of this transition, transmission electron

128

microscopy (TEM) was performed on HaCaT cells infected with 30 MOI

M. globosa

at 0, 0.5, 4, and 24

129

hours post-infection. Infected keratinocytes exhibited distinct time-dependent morphological alterations:

130

(1) Desmosome formation and extracellular vesicles were dramatically escalatory at 0.5 hours, suggestive

131

of the initiation of immediate cellular defense; (2) Secondary lysosomes and electron-dense lipid droplets

132

were prominent at 4 hours, indicative of active metabolic and phagocytic responses; (3) Mitochondrial

133

structures were severely damaged at 24 hours, such as complete disintegration of the outer membrane,

134

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dissolution of inner cristae into monolayered annular structures, and dispersion of inner membrane

135

remnants in the cytoplasm (Figure 3A).

136

Concurrently, some

M. globosa

spores entered HaCaT cells at 4 hours post-infection. Intracellular spores

137

exhibited nuclear chromatin condensation and increased electron density; intravacuolar spores remained

138

spherical with attenuated cell wall staining; and extracellular spores were polymorphic, with some partially

139

engulfed by keratinocyte pseudopodia through undulated electron-dense contact zones. At 24 hours,

140

intracellular spores underwent further degradation, releasing fine electron-dense granules, some of which

141

escaped from necrotic keratinocytes. However, extracellular spores retained a morphology similar to that

142

of intravacuolar spores at 4 hours (Figure 3B). Collectively, these ultrastructural alterations were

143

progressive and conspicuous at 24 hours post-infection, suggesting the underlying interaction between

144

globosa

and keratinocytes during infection.

145

Activation of PAR2

–β-arrestin 2–ERK signaling axis and IL-17 in M. globosa-infected HaCaT cells

146

The PAR2 signaling pathway has been shown to function through a

β-arrestin-mediated mechanism that

147

is independent of G protein coupling.

48,49

The

β-arrestin acts as a critical regulator, not only terminating G

148

protein signaling but also facilitating PAR2 internalization and scaffolding the formation of the PARs

–β-

149

arrestin

–Raf-1–MEK-1–ERK1/2 complex. This process prolongs cytosolic ERK1/2 activity by delaying its

150

nuclear translocation.

Given PAR2’s role in IL-17 regulation across various diseases,

51,52

151

investigated its involvement in

M. globosa

-induced IL-17 production in HaCaT cells. Our temporal

152

analysis revealed that

M. globosa

infection significantly triggered IL-17 release at 12 hours post-infection.

153

Western blotting and qRT-PCR correlation analysis indicated that IL-17 production was mediated through

154

PAR2 activation, with

potential involvement of β-arrestin 2, ERK2, and TJ protein ZO-1 (Figure 2B-F).

155

Based on previous findings that PAR2 enhances intestinal barrier function through β-arrestin–ERK1/2-

156

dependent upregulation of TJs,

we employed pharmacological and genetic approaches to delineate this

157

pathway in keratinocytes. HaCaT cells were treated

with 50 μM PAR2 antagonist FSLLRY-NH2

53,54

for 1

158

hour (Figure S3), followed by infection with 30 MOI

M. globosa

at 24 hours. Concurrently, HaCaT cells

159

were treated with 10 μM PAR2 agonist SLIGRL-NH2 for 24 hours. The PAR2 antagonist reversed

160

globosa

induced upregulation of β-arrestin 2, phospho-ERK, IL-17, and Occludin, as well as the

161

downregulation of ZO-1 in HaCaT cells (Figure 4A, B). PAR2-specific siRNA abolished

M. globosa-

162

induced increases in β-arrestin 2, p-ERK, and IL-17, while β-arrestin 2 siRNA blocked the elevation of p-

163

ERK and IL-17. Both treatments normalized ZO-1 and IL-17 expression (Figure 4C-E).

164

Notably, although

M. globosa

infection caused significant mitochondrial damages of HaCaT cells,

165

including outer membrane disintegration and cristae dissolution, the PAR2 antagonist FSLLRY-NH2

166

effectively prevented

M. globosa

-induced loss of mitochondrial membrane potential (Figure 4F, G). These

167

collective findings indicate that PAR2

–β-arrestin 2–ERK axis may be pivotal in

M. globosa

-induced IL-17

168

release in HaCaT cells.

169

PAR2 interacts with β-arrestin 2 and ZO-1 in M. globosa-infected HaCaT cells

170

To elucidate the functional interplay between PAR2, β-arrestin 2, and ZO-1 during

M. globosa

infection,

171

HaCaT cells were pretreated with the PAR2 antagonist FSLLRY-

NH2 (50 μM) for 1 hour, followed by

172

infection with

M. globosa

(30 MOI) for 24 hours. Immunofluorescence and PLA tests revealed enhancive

173

co-

localizations of PAR2 with both β-arrestin 2 and ZO-1 upon

M. globosa

infection or stimulation with

174

PAR2 agonist SLIGRL-NH2 (Figure 5A-D). Co-IP assays further confirmed that

M. globosa

infection and

175

PAR2 agonist pretreatment significantly strengthened the physical interaction among PAR2, ZO-

1, and β-

176

arrestin 2, which was effectively attenuated by PAR2 antagonist (Figure 5E, F).

177

To independently validate these protein interactions, full-length coding sequences of PAR2, ZO-1,

and β-

178

arrestin 2 were cloned into plasmid vectors and expressed in the yeast two-hybrid strain NMY51.

179

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Screening results identified molecular interactions among PAR2, ZO-

1, and β-arrestin 2 (Figure 5G),

180

supporting the role of PAR2 as a scaffold protein that facilitates complex formation in response to

181

globosa

challenge.

182

Attenuation of PAR2

–β-arrestin 2–ERK signaling and IL-17 responses in Par2

-/-

mice with skin M.

183

globosa infection

184

To evaluate the role of the PAR2

–β-arrestin 2–ERK signaling axis and IL-17 in

Malassezia

infection

185

vivo

, we established a cutaneous

M. globosa

infection model in mice, as previously described.

17,24

This

186

model was designed to delineate the mechanism by which

M. globosa

induces IL-17 production in

187

keratinocytes via the PAR2

–β-arrestin 2–ERK signaling axis. Twelve

Par2

-/-

or wild-type (WT) mice were

188

randomly assigned to Vehicle and

M. globosa

groups. Animals were euthanized on day 7 post-infection.

189

In WT mice, infection

with

M. globosa

induced remarkable clinical and histopathological changes by day

190

7, including scaling, crusting, epidermal hyperplasia, spongiosis, and dermal vascular dilation with

191

lymphohistiocyte infiltration(Figure 6A). In contrast, these clinicopathological manifestations were

192

markedly attenuated in

Par2

-/-

mice. IHC results showed that the expression trends of PAR2, IL-17, and

193

ZO-1 in WT mice infected with

M. globosa

were similar to those observed in MF, whereas

Par2

-/-

mice

194

were not affected by

M. globosa

infection. These findings suggest that PAR2 plays a crucial regulatory

195

role in the pathogenesis of MF(Figure 6B, C).

196

Immunofluorescence analysis revealed prominent co-

localization of PAR2 with β-arrestin 2 and ZO-1 in

197

WT mice following infection, which was absent in

Par2

-/-

mice (Figure 6D, E). Furthermore, infection-

198

induced changes in the

expression and distribution of PAR2, β-arrestin 2, and ZO-1 were evident in WT

199

mice but were negligible in

Par2

-/-

mutants. Western blotting found that

M. globosa

infection upregulated

200

PAR2, β-arrestin 2, p-ERK, and IL-17 expression in WT mice but not in

Par2

-/-

mice. Downexpression of

201

ZO-1 was observed in both genotypes, particularly in WT mice (Figure 6F, G). ELISA results indicated

202

that cutaneous IL-17 levels was elevated in

M. globosa

-infected WT mice and minimal in

Par2

-/-

mice

203

(Figure 6H).

204

Collectively, these in vivo results substantiate the involvement of the PAR2

–β-arrestin 2–ERK axis in

205

globosa-

induced cutaneous inflammation and IL-17 production.

206

DISCUSSION

207

Previous studies have established the role of Th17 immunity in

Malassezia

infection and other fungal

208

infections of mouse skin.

18,19,24,25,55

However, the mechanism by which

Malassezia

induces an IL-17

209

response in keratinocytes remains unclear. As a major component of the epidermis, keratinocytes not

210

only provide a physical barrier but also initiate immune responses by recruiting and activating immune

211

cells, thereby triggering local inflammation.

56,57

Malassezia

infection triggers the coordinated antifungal

212

immunity mediated by the IL-17 signaling pathway in keratinocytes, which promotes cell migration and

213

exacerbates cutaneous inflammation.

15,18,19,24

Our results demonstrate the presence of an IL-17 immune

214

response in MF lesions,

M. globosa

-infected HaCaT cells, and mouse skin, consistent with previous

215

studies.

216

The proteases secreted by

M. globosa

function as major virulence factors, promoting nutrient acquisition

217

through proteolysis and degradation of host tissues, facilitating adhesion by modifying host cells,

218

regulating immune responses via secretion of proinflammatory cytokines, and being directly recognized

219

by host membrane-bound PRRs.

58-63

This study identified that PAR2, a non-canonical PRR, exhibited

220

time-dependent upregulation during the infection process in

M. globosa

-infected HaCaT cells and was

221

positively correlated with IL-17 levels. Serine protease is the primary protease secreted by

M. globosa

222

and acts as a key activator of PAR2.

9,64

The intracellular endocytosis of PAR2 depends on its interaction

223

with β-arrestin, which is crucial for the targeted regulation of ERK1/2.

50,65

Inhibition of the PAR2

–β-

224

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arrestin

–ERK1/2 axis has been shown to overcome osimertinib resistance in non-small cell lung cancer,

225

and this signaling pathway can also regulate intestinal inflammation by modulating mucosal barrier

226

integrity through TJ proteins of epithelial cells.

Although PAR2 mediates various physiological and

227

pathological processes via the

β-arrestin 2–ERK axis, its role in

M. globosa

infection remains unreported.

228

In both cellular and mouse infection models, we observed that PAR2 inhibition or knockout restored the

229

down

regulation of TJ proteins, upregulation of β-arrestin 2–ERK1/2 axis, and IL-17-driven inflammatory

230

response in

M. globosa

infected keratinocytes. Further studies revealed that PAR2 interacted with β-

231

arrestin 2 and ZO-1 to regulate the inflammatory response. Interestingly, we found that ZO-1, Occludin,

232

and Claudin-1 were all downregulated in MF, but only ZO-1 was restored by PAR2 intervention, indicating

233

that PAR2 regulates ZO-1 expression and interacts with ZO-1 to modulate the infection process. This

234

differential regulation may be related to the structural and functional characteristics of ZO-1, Occludin,

235

and Claudin-1. PAR2-regulated TJs are believed to participate in the pathogenesis of chronic

236

inflammatory diseases. For instance, PAR2 downregulates ZO-1 and Claudin-1 expression, contributing

237

to the epithelial barrier dysfunction in allergic rhinitis.

TJs are composed of transmembrane proteins and

238

plaque proteins: transmembrane proteins include Claudins, TJ-related MARVEL proteins (such as

239

Occludin, Tricellulin, MarvelD3), and junction adhesion molecules (JAM); plaque proteins include Cingulin

240

(ZO) 1-3, MUPP-1 and Cingulin.

PAR2 activation leads to TJ disruption in epithelial cells and increases

241

barrier permeability through p38 MAPK activation.

In contrast, treatment with

Pseudomonas aeruginosa

242

elastase temporarily disrupts the epithelial barrier, downregulated transmembrane proteins Claudin-1 and

243

-4, Occludin, and Tricellulin, but does not downregulate scaffold proteins ZO-1 and -2 or adhesion

244

junction proteins E-

cadherin and β-catenin.

Our results indicate that PAR2 regulates the scaffold protein

245

ZO-1 in

Malassezia

-infected keratinocytes, while downregulation of membrane proteins Occludin and

246

Claudin-1 may be directly related to the destructive effect of

Malassezia

proteases. Additionally, using

247

TEM and JC-1 staining, we first demonstrated that

M. globosa

infection disrupts the mitochondrial

248

structure and membrane potential of HaCaT cells, an effect reversed by PAR2 inhibitor. The PAR2

249

inhibitor AZ3451 can significantly improve mitochondrial function by increasing intracellular adenosine

250

triphosphate (ATP) levels, restoring mitochondrial membrane potential, and reducing mitochondrial

251

ROS.

71-73

The specific mechanism by which PAR2 regulates mitochondria function in

Malassezia

-infected

252

HaCaT cells requires further investigation.

253

In conclusion, this study demonstrates that PAR2 disrupts ZO-1 to mediate IL-17-driven inflammatory

254

response in

M. globosa

-infected HaCaT cells and mouse skin via activation of the

β-arrestin 2–ERK

255

signaling pathway (Figure 7). Thus, PAR2 participates in the pathogenesis of

Malassezia

-related skin

256

diseases and may represent a promising therapeutic target.

257

258

Limitations of the study

259

In this study, we demonstrated that

M. globosa

activated the PAR2

–β-arrestin 2–ERK signaling pathway

260

in HaCaT cells and mouse skin, thereby mediating IL-17-driven inflammatory response. However, this

261

effect remains to be testified using NHEK and skin organoids. Meanwhile, the specific functional

262

interaction regions and mechanisms of PAR2/β-arrestin 2/ZO-1 need to be verified. In addition, the role of

263

PAR2 in the transition between commensal and pathogenic

Malassezia

is well worth investigating.

264

265

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RESOURCE AVAILABILITY

266

Lead contact

267

Further information and requests for resources and reagents should be directed to and will be fulfilled by

268

the Lead Contact, Yi-Ming Fan (ymfan1963@163.com).

269

Materials availability

270

Materials used in the study are commercially available. This study did not generate new unique reagents.

271

Data and code availability

272

All data reported in this paper will be shared by the lead contact upon reasonable request.

273

Proteomics data of MF have been deposited at Science Data Bank and are publicly available as of the

274

date of publication at https://doi.org/10.57760/sciencedb.29934 (listed in the key resources table). Raw

275

data have been deposited at Mendelay Data (https://data.mendeley.com/datasets/ffxppsr7p2/1)

276

No new code was generated during the course of this study.

277

Any addition information required to reanalyze the data reported in this paper is available from the lead

278

contact upon reasonable request.

279

ACKNOWLEDGMENTS

280

We thank Nicola Lopizzo for scientific editing. We thank Applied protein technology and Shenzhe biotech

281

for excellent technical support. This work was supported by the National Natural Science Foundation of

282

China (grant No. 81673071, 82404162).

283

AUTHOR CONTRIBUTIONS

284

Conceptualization, Y.M.F. and J.F.T.; methodology, J.Q.H., X.Y.L., and J.L.; Investigation, J.F.T., F.G.L.,

285

J.Q.H., and Y.Z.; writing

—original draft, J.F.T. and F.G.L.; writing—review & editing, Y.M.F and Y.Z.;

286

funding acquisition, Y.M.F. and J.L.; resources, J.F.T. and F.G.L.; supervision, Y.M.F.

287

DECLARATION OF INTERESTS

288

The authors declare no competing interests.

289

290

291

292

293

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FIGURE TITLES AND LEGENDS

299

Figure 1. Expression of PAR2, IL-17 and tight junction (TJ) proteins in Malassezia folliculitis

300

(A, B) PAR2, IL-17, ZO-1, Occludin, and Claudin-1 were validated in MF and CTRL skin tissue by

301

Immunohistochemical staining(IHC). Values are mean ± SD of six samples from two independent

302

experiments. Statistical analysis was performed using a two-

way ANOVA with Šídák’s multiple

303

comparisons test (*

< 0.05; ***

< 0.001).

304

(C, D) The Spores rate of CTRL and MF groups by PeriodicAcid

–Schif staining. Purple arrows indicate

305

he spores. Scale bar: 50 μm.. Data are presented as mean ± SD of triplicate wells from three

306

independent experiments. Statistical significance was determined using unpaired two-

tailed Student’s t-

307

tests (**

< 0.01).

308

Figure 2. Impact of M. globosa infection on PAR2 activation, IL-17 production and TJ integrity in

309

HaCaT cells

310

(A) Cell survival rate was detected by CCK8 assay.

311

(B, C, D) Protein expression and correiation analysis in HaCaT cells infected with 30 MOI

M. globosa

was

312

evaluated by Western blot at various time.

313

(E, F) mRNA levels and correiation analysis were measured by qRT-PCR. Red indicated up-regulated

314

gene expression, green indicated down-regulated gene expression.

315

(G) IL-17A levels were detected by ELISA test in the supernatant of HaCaT cells infected with 30 MOI

316

globosa

. Red indicated up-regulated gene expression (C, E) or positive correlation(D, F), blue indicated

317

down-regulated gene expression (C, E) or negative correlation (D, F).These experiments (A, B, E, G)

318

were representative of 3 independent experiments. Values are means ± SD. a two-way ANOVA with

319

Bonferroni’s (A), or Dunnet’s (G) multiple comparisons test was applied. *

< 0.05, **

< 0.01, ***

320

0.001. Correlation analysis is performed using Pearson correlation coefficient analysis. See also Figure

321

S2.

322

Figure 3. Ultrastructural alterations of HaCaT cells and M. globosa interaction during infection

323

(A) Ultrastructural alterations of cells. Red arrows denote lysosomes or autophagolysosomes, light blue

324

arrows denote desmosome-like structures, dark blue arrows denote lipid droplets, green arrows denote

325

extracellular vesicles, and yellow arrows denote mitochondria.

326

(B) Ultrastructural changes of intracellular and extracellular spores. Purple arrows indicate intracellular

327

spores. Scale bars: 2 μm, 1 μm, 500 nm. Three biological replicates were conducted. Representative data

328

are presented.

329

Figure 4. Activation of PAR2

–β-arrestin 2–ERK signaling axis and IL-17 in M. globosa-infected

330

HaCaT cells

331

(A, B) Protein expression level following PAR2 antagonist intervention using Western blotting.

332

(C, D) PAR2-related pathway proteins expression following siRNA transfection by Western blotting.

333

(E) IL-17 levels in cell supernatants after siRNA transfection by ELISA.

334

(F, G) Fluorescence of JC-

1 aggregates to monomers. Scale bar: 10 μm. These experiments (A, C, E, F)

335

were representative of 3 independent experiments. Values are mean ± SD. Statistical analysis was

336

performed using a two-

way ANOVA with Šídák’s multiple comparisons test (ns: not satisticant; *

< 0.05;

337

< 0.01; ***

< 0.001). See also Figure S3.

338

Figure 5

. PAR2 interacts with β-arrestin 2 and ZO-1 in M. globosa-infected HaCaT cells

339

(A, B) Eexpression and co-

localization of PAR2, β-arrestin 2, and ZO-1 in HaCaT cells by

340

immunofluorescence. White arrows: co-

localization. Scale bar: 10 μm.

341

Journal Pre-proof

(C, D) Expression and co-localization of PAR2 and ZO-1 by PLA. The yellow arrows indicated the PLA-

342

positive signal, Scale bar: 20 μm.

343

(E, F) Interaction regulation of PAR2, β-arrestin 2 and ZO-1 using Co-IP. These experiments (A, C, E)

344

were representative of three independent experiments. Data are presented as mean ± SD. Statistical

345

analysis was performed using a two-

way ANOVA with Šídák’s multiple comparisons test (ns: no satistical

346

significance; *

< 0.05; **

< 0.01; ***

< 0.001).

347

(G) PAR2 interaction with ZO-

1 and β-arrestin 2 using MbYTH. G1. MbYTH of PAR2 and control NR1I2.

348

a. Positive control, b. Negative control, c. Experimental group: pDHB1-PAR2 and pPR3-N-NR1I2, d.

349

Autoactivation 1: pDHB1-PAR2, e. Autoactivation 2: pPR3-N-NR1I2. G2. MbYTH of PAR2 and ZO-1. a.

350

Positive control; b. Negative control; c. Experimental group: pDHB1-PAR2 and pPR3-N-ZO-1; d.

351

Autoactivation 1: pDHB1-PAR2; e. Autoactivating2: pPR3-N-ZO-

1. G3. MbYTH of PAR2 and β-arrestin 2.

352

a. Positive control; b. Negative control; c. Experimental group: pDHB1-PAR2 and pPR3-N-

β-arrestin 2; d.

353

Autoactivation: pPR3-N-

β-arrestin 2. SD/-Leu/-Trp medium; SD/-Leu/-Trp/-His medium containing 5mM 3-

354

AT; SD/-Leu/-Trp/-His/-Ade medium; SD/-Leu/-Trp/-His/-Ade medium containing 5mM 3-AT. 1/2/3/4 were

355

10000/1000/100/10 times dilution of the original bacterial solution.

356

Figure 6. Attenuation of PAR2

–β-arrestin 2–ERK signaling and IL-17 responses in Par2

-/-

mice with

357

skin M. globosa infection

358

(A) Skin lesions and pathological staining of mouse skin. Scale bar: 50 μm.

359

(B, C) Expression level of PAR2, IL-17, ZO-1 by IHC.

360

(D, E) Expression and co-

localization of PAR2, β-Arrestin 2, and ZO-1 by immunofluorescence. White

361

dotted line indicated the basement membrane zone. Scale bar: 50 μm.

362

(F, G) Expression of related proteins using Western blotting.

363

(H) IL-17 levels using ELISA. (B, C, H)n = 6 for each group. These experiments (A, D, E, F, G) were

364

representative of 3 independent experiments. Data are presented as mean ± SD. Statistical significance

365

was determined using a two-way ANOVA with

ídák

’

s multiple comparisons test (ns: no satistical

366

significance; *

< 0.05; **

< 0.01; ***

< 0.001).

367

Figure 7. The possible mechanism of PAR2

–β-arrestin 2–ERK signaling pathway on IL-17 release

368

in keratinocytes

369

On physiological state, PAR2, β-arrestin 2 and ZO-1 are in resting state, and ZO-1 is crucial for skin

370

barrier maintenance. After overgrown

M. globosa

invades keratinocytes, the interaction of PAR2 with β-

371

arrestin 2 and ZO-1 is enhanced and internalized, which upregulates ERK1/2 expression and ZO-1

372

disruption, thus promoting intracellular expression and extracellular release of IL-17.

373

374

375

376

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377

378

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interactions using the split-ubiquitin membrane yeast two-hybrid system. Methods Mol. Biol.

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670

225-44. http://doi.org/10.1007/978-1-61779-455-1_13.

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92. Iyer, K., Bürkle, L., Auerbach, D., Thaminy, S., Dinkel, M., Engels, K., and Stagljar, I. (2005). Utilizing

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673

integral membrane proteins. Sci. STKE.

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674

675

Journal Pre-proof

STAR

★

METHODS

676

KEY RESOURCES TABLE

677

678

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

679

MF lesions and normal skin

680

Nine MF lesions and normal skin (CTRL) were collected from the Department of Dermatology, Affiliated

681

Hospital of Guangdong Medical University. The study was approved by the Hospital's Ethics Committee

682

(No. PJKT2024-193). Inclusion criteria: (1) Typical manifestations of MF; (2) Calcofluor white stain of skin

683

scrapings showing

Malassezia

spores and/or hyphae; (3) No systemic antifungal drugs, glucocorticoids or

684

immunosuppressants within one month and no topical antifungal drugs within two weeks; (4) No

685

congenital and acquired immunodeficiency.

686

Cell lines and culture conditions

687

HaCaT cells (cat#iCell-h066) were purchased from the iCell Bioscience Inc. (Shanghai, China) and

688

cultured in high-glucose Dulbecco's modified Eagle medium (Gibco, NY, USA) with 10% fetal bovine

689

serum (Gibco) at 37°C in 5% CO

.The HaCaT cells have been authenticated by iCell Bioscience Inc.,

690

using a 21-STR amplification protocal. The cells were no mycoplasma contamination as tested by TEM.

691

Fungal culture

692

M. globosa

type strain CBS9597 was obtained from the Institute of Dermatology, Chinese Academy of

693

Medical Sciences and Peking Union Medical College (Nanjing, China).

The yeasts were cultivated in

694

modified Leeming-Notman broth for 4-

5 days at 32°C, then harvested by centrifugation, washed thrice in

695

PBS and diluted in medium.

696

Animals

697

Par2

-/-

and wild-type (WT) C57BL/6 mice were purchased from GenePharma (Jiangsu, China). The

698

identification profiles for

Par2

-/-

and WT mice are provided in Figure S5. Genotype mice were indentified

699

using two pairs of primers(Primers

①

: Forward, 5’-CTCAGTGGGAGGATGTTTATGAAG-3’, Reverse, 5’-

700

CTGACCGTTCTATTCCAGAAATACAG-

3’, Band size: WT: 4100 bp,

Par2

-/-

: 268 bp

；

Primers

②

701

Forward, 5’-CTCAGTGGGAGGATGTTTATGAAG-3’, Reverse, 5’-CAACCACCAGCAGTCTTTGATTG-3’,

702

Band size: WT: 380 bp,

Par2

-/-

: 0 bp)(Figure S4). The PCR program was as follows: 95

℃

for 5 min; 98

℃

703

for 30 s, 65

℃

for 30 s,72

℃

for 45 s (20 cycles); 98

℃

for 30 s, 55

℃

for 30 s,72

℃

for 45 s (20 cycles);

704

℃

for 5 min, 10

℃

hold. The animal experiments were approved by the Affiliated Hospital's Ethics

705

Committee of Guangdong Medical University (No. AHGDMU-LAC-

Ⅱ

(1)-2302-A055). Littermates of the

706

same sex were randomly assigned to experimental groups, and the breeding was conducted in the SPF-

707

grade animal breeding facility of Guangdong Medical University Affiliated Hospital. Previous multiple

708

studies

9,74 ,75

by our research group have demonstrated that males are more susceptible to MF than

709

females, highlighting a gender difference. Consequently, subsequent studies used male mice as the

710

infection model. Male mice aged 6-

8 weeks were chosen.

M. globosa

-infected mouse skin models were

711

established as previous reports.

17,18

Mice were anesthetized by intraperitoneal injection of 1%

712

pentobarbital sodium. Their dorsal skin was stripped once using 3M Durapore Surgical Tape (3M Health

713

Care, MN, USA) after hair shaving, and then treated topically with 200 μL of 10

spores/mL

M. globosa

714

glycerol once daily for seven consecutive days.

715

716

METHOD DETAILS

717

LC-MS/MS analysis

718

Three MF lesions and normal skin were collected from the Department of Dermatology, Affiliated Hospital

719

of Guangdong Medical University. SDT (4%SDS, 100mM Tris-HCl, pH7.6) buffer was used for sample

720

Journal Pre-proof

lysis and protein extraction. The amount of protein was quantified with the BCA Protein Assay Kit

721

(Beyotime, Shanghai, China).The proteins were separated on SDS-PAGE gel (constant voltage 180V, 45

722

min). Protein digestion by trypsin was performed according to filter-aided sample preparation (FASP)

723

procedure described by Matthias Mann.

The digest peptides of each sample were desalted on C18

724

Cartridges (Sigma-Aldrich, MI, USA). LC-MS/MS analysis was performed on a timsTOF Pro mass

725

spectrometry (Bruker, Karlsruhe, Germany) that was coupled to Evosep One system liquid

726

chromatography (Evosep, Odense, Denmark). Finally, the MS data of samples were analyzed by

727

bioinformatics as previous reports.

77,78

728

Cell viability and ELISA

729

Cell viability was determined with the Cell Counting Kit-8 (CCK8, Beyotime, Shanghai, China), following

730

the manufacturer’s protocol. HaCaT cells were plated in 96-well plates at a density of 3,000 cells per well

731

and allowed to adhere for 24 h before treatment. Subsequently, 10 μL of CCK-8 solution was added to

732

each well, and after 2 h of incubation, the absorbance at 450 nm was measured using a microplate reader.

733

Interleukin-17 (IL-17) levels were quantified using commercial enzyme-linked immunosorbent assay

734

(ELISA) kits (Me

imian Industrial, Jiangsu, China) in strict adherence to the manufacturer’s instructions.

735

Cell culture supernatants, tissue culture supernatants, and serially diluted standards were coated onto

736

microplate wells. Following a washing step, the wells were blocked and then incubated with an anti-IL-17

737

primary antibody at 37°C for 2 h. After further washing, the wells were incubated with a horseradish

738

peroxidase (HRP)-conjugated secondary antibody, washed again, and developed with substrate. The

739

reaction was sto

pped, and absorbance at 450 nm was recorded using a microplate reader.

740

Quantitative Real-Time (qRT) - PCR

741

qRT-PCR was performed as previously described.

Total RNA was extracted using TRIzol reagent

742

(Thermo Fisher Scientific, USA) and reverse transcribed using

Evo M-MLV

reverse transcription Kit

743

(Aikori Biological Engineering CO., Ltd, Changsha, China). mRNA expression was quantified via the

744

SYBR green method (Aikori Biological Engineering CO., Ltd, Changsha, China) on a reverse transcription

745

machine (LC480, USA). Specific primers of PAR2, β-arrestin 2, EKR1, ERK2, IL-17, ZO-1, Occludin,

746

Claudin-1 and GAPDH were synthetized by Sangon Biotech (Shanghai, China). All samples were

747

normalized to endogenous controls and fold changes calculated relative to control. The control GAPDH

748

was used for normalization, and relative mRNA levels were calculated using the 2

△△

method.

749

Western Blotting analysis

750

The protein fractions were extracted using Protein Extraction Kit (Beyotime, Shanghai, China). Based on

751

our previous experiment,

the blots were incubated with primary antibodies PAR2, β-arrestin 2, p-ERK,

752

ERK1/2, IL-17, ZO-1, Occludin, Claudin-1, and GAPDH at a 1:1000 dilution. They were then incubated

753

with HRP-conjugated secondary antibodies (Proteintech, Wuhan, China) and visualized using enhanced

754

chemiluminescence (Millipore, MA, USA). All experiments were conducted with three independent

755

repetitions.

756

Immunohistochemical staining (IHC)

757

Immunostaining for PAR2, IL-17, ZO-1, Occludin, and Claudin-1 was performed on paraffin-embedded

758

sections as previously described.

Following antigen retrieval and blocking, paraffin-embedded sections

759

were incubated with primary antibodies at 4°C overnight, after which they were treated with

760

corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies. Chromogenic detection

761

was carried out using diaminobenzidine (DAB), and images were acquired using light microscopy.

762

Immunofluorescent staining (IF)

763

HaCaT cells were cultured in confocal Petri dishes and treated with 30 MOI

M. globosa

for 24 hours.

764

Immunofluorescence staining for PAR2, β-arrestin 2, and ZO-1 was conducted as previously described.

765

Specimens (tissues or cells) were fixed in 4% paraformaldehyde (Solarbio, Beijing, China) for 30 minutes,

766

Journal Pre-proof

followed by permeabilization and blocking. The samples were then incubated with appropriate primary

767

antibodies, followed by a light-protected incubation with fluorophore-conjugated secondary antibodies.

768

Nuclear staining was performed using DAPI, and slips were mounted with an anti-fade medium. Image

769

acquisition and observation were carried out on a laser scanning confocal microscope.

770

Periodic acid-Schiff staining(PAS)

771

The experimental protocol was conducted following the manufacturer’s instructions for the Periodic Acid

772

Schiff (PAS) Stain Kit (Solarbio, Beijing, China). Paraffin-embedded tissue sections were deparaffinized in

773

xylene and gradually rehydrated through a graded ethanol series. After oxidation in periodic acid solution,

774

the sections were incubated with Schiff’s reagent at 30 °C for 45 min. Nuclei were counterstained with

775

hematoxylin for 30 s, followed by differentiation in acid-alcohol and bluing in running tap water for 5 min.

776

Finally, the sections were dehydrated, cleared, mounted, and imaged using light microscopy.

777

siRNA transfection

778

PAR2 a

nd β-arrestin 2-targeted and scrambled siRNAs were designed by GenePharma (Shanghai,

779

China). Briefly, adherent HaCaT cells were transfected with these siRNAs for 6-

8 hours using

780

Lipofectamin 3000 reagents and Opti-MEM (Thermo Fisher Scientific, CA, USA). Following transfection,

781

the cells were cultured in fresh medium and co-cultured with 30 MOI

M. globosa

for 24 hours. The

782

knockdown efficiency was verified by qRT-PCR and western blotting.

783

Transmission electron microscopy (TEM)

784

HaCaT cells were cultured on pre-sterilized Aclar film (Ted Pella, CA, USA) or 6-well cell cultrure

785

plate(Nest, Jiangsu, China), treated according to the experimental settings. The samples were fixed with

786

3% glutaraldehyde and 1% osmium tetroxide, followed by gradient ethanol dehydration. They were then

787

vacuum-infiltrated with ethanol or acetone resin using a microwave treatment instrument (Ted Pella, CA,

788

USA) and polymerized in pure resin. After top buckle embedding, the blocks were trimmed and sectioned.

789

The 70 nm ultrathin sections were double-stained with lead citrate and uranyl acetate, and observed at 80

790

kV on the JEM1400 (JEOL Ltd., Tokyo, Japan) for photography. The method is cited as previously

791

described.

81-83

792

Co-immunoprecipitation (Co-IP)

793

Cell lysates were extracted using Western and IP cell lysis buffer (Beyotime, Shanghai, China). Following

794

centrifugation, the supernatants were incubated overnight with Protein A/G Magnetic Beads (MCE,

795

Shanghai, China) and IP antibodies. The beads were then washed four times with the same lysis buffer,

796

resuspended in 40 μL of 1% SDS-PAGE sample loading buffer, and boiled at 95°C for 5 minutes. The

797

method is detailed as previously described.

84-86

798

Proximity ligation assay (PLA)

799

HaCaT cells were cultured on sterile slides and prepared according to the experimental design. The PLA

800

method is cited as previously described.

87-89

The cells were fixed with 4% paraformaldehyde for 30

801

minutes, followed by treatment with Duolink

blocking solution (Sigma-Aldrich, MI, USA). Two species-

802

specific primary antibodies were added and incubated at 4°C overnight. After incubation with Duolink

803

PLA probes, cells proceeded sequentially with Duolink

ligase for ligation and Duolink

polymerase for

804

amplification. Nuclei were stained using DAPI-containing Duolink

in situ sealing agent (Sigma-Aldrich,

805

MI, USA). Cells were then observed and analyzed with an LSM900 laser confocal microscope (ZEISS,

806

Jena, Germany). A negative control without primary antibodies was included.

807

Membrane-based yeast two-hybrid (MbYTH)

808

After constructing the plasmid vector for the target gene using pDHB1 or pPR3-N (Figure S1, Table S1),

809

NMY51 yeast (LSM Bio, Wuhan, China) competent cells were prepared using LiAc method. Then, the

810

plasmid vectors were transfected into the competent NMY51 yeast. The clones were successively

811

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screened using SD/-Leu/-Trp, SD/-Leu/-Trp/-His with 5 mM 3-AT, SD/-Leu/-Trp/-His/-Ade, and SD/-Leu/-

812

Trp/-His/-Ade with 5 mM 3-AT. The continuously growing clones indicated the presence of protein

813

interactions; otherwise, it would be a false positive result. The experimental groups were detailed in Table

814

S2. The method reference remains as previously mentioned.

90-91

815

QUANTIFICATION AND STATISTICAL ANALYSIS

816

Statistical analyses were performed using GraphPad Prism software (version 9.4.0). Comparisons

817

between two groups were made using unpaired two-

tailed Student’s t-tests (Figure 1B, 1D, 6C, 6D, 6G,

818

and 6H). For datasets containing one independent variable in multiple groups, a one-way ANOVA

819

followed by Dunnet’s multiple comparisons test was performed (Figures 2G and S3C). For multiple group

820

comparisons, a two-way ANOVA

with Bonferroni’s (Figure 2A, S3B, S3D), or Šídák’s (Figures 4B, 4D, 4E,

821

4G, 5B, 5D, 5F, and S3A) multiple comparisons test was applied. *

< 0.05, **

< 0.01, ***

< 0.001, and

822

▲▲▲

< 0.001(Figure S3B) were considered statistically significant.

A Pearson’s correlation analysis

823

was conducted, and the values represent the correlation coefficient (Figure 2D, 2F).

824

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KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

Rabbit monoclonal anti-PAR2

Abcam

Cat#ab180953;
RRID:AB_3674622

Goat polycional anti-

β-arrestin 2

Abcam

Cat#ab31294;
RRID:AB_2060265

Mouse monoclonal anti-p-ERK

Santa cruz

Cat#sc-7383;
RRID:AB_627545

Mouse monoclonal anti-ERK1/2

Santa cruz

Cat#sc-514302;
RRID:AB_2571739

Rabbit polyclonal anti-IL-17

Affinity

Cat#DF6127;
RRID:AB_2838094

Rabbit polyclonal anti-ZO-1

Thermo Fisher
Scientific

Cat#61-7300;
RRID:AB_138452

Mouse monoclonal anti-Occludin

Thermo Fisher
Scientific

Cat#33-1500;
RRID:AB_2533101

Mouse monoclonal anti-Claudin-1

Thermo Fisher
Scientific

Cat#37-4900;
RRID:AB_2533323

Mouse monoclonal anti-GAPDH

Proteintech

Cat#CL594-
60004;RRID:AB_291
9886

Alexa Fluor 555-labeled donkey anti-rabbit IgG

Beyotime

Cat#A0453;
RRID:AB_2890132

Alexa Fluor 647-labeled Goat Anti-Mouse IgG

Beyotime

Cat#A0473;
RRID:AB_2891322

Dylight 488-labeled donkey anti-goat IgG

Abcam

Cat#ab96935,
RRID:AB_10679538

Rabbit anti-goat IgG-HRP

Santa cruz

Cat#sc-2768,
RRID:AB_656964

Goat anti-mouse IgG-HRP

Santa cruz

Cat#sc-2005,
RRID:AB_631736

Goat anti-rabbit IgG-HRP

Proteintech

Cat#SA00001-2,
RRID:AB_2722564

Bacterial and virus strains

M. globose

type strain CBS9597

Luo et al.

Error! Reference

source not found.

N/A

NMY51 yeast

LSM Biological

Cat#S0094

Biological samples

Healthy adult skin tissue

This paper

N/A

Human MF lesions

This paper

N/A

Infected mouse skin tissue

This paper

N/A

Chemicals, peptides, and recombinant proteins

PAR2 antagonist FSLLRY-NH2

Sigma-Aldrich

Cat#SML0714

PAR2 agonist SLIGRL-NH2

Sigma-Aldrich

Cat#S9317

Lipofectamine(RNAiMAX transfection reagent)

Thermo Fisher
Scientific

Cat#13778150

Critical commercial assays

TRIzol reagent

Thermo Fisher
Scientific

Cat#12183018A

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Evo M-MLV

reverse transcription Kit

Aikori Biological

Cat#AG11705

SYBR Green

Pro Taq

HS Pre-mixed qPCR Kit (with

ROX)

Aikori Biological

Cat#AG11718

Duolink® In Situ red test reagent

Sigma-Aldrich

Cat#DUO92008

Duolink® In-situ PLA® probe anti-Rabbit PLUS

Sigma-Aldrich

Cat#DUO92002

Duolink® in-situ PLA® probe anti-mouse MINUS

Sigma-Aldrich

Cat#DUO92004

Duolink® in-situ PLA® detection mounting medium,
containing DAPI

Sigma-Aldrich

Cat#DUO82040

Deposited data

Proteomics data of MF

This paper, Science
Data Bank

https://doi.org/10.57
760/sciencedb.2993
4

Raw images

This study

https://data.mendele
y.com/datasets/ffxpp
sr7p2/1

Experimental models: Cell lines

HaCaT cells

iCell Bioscience Inc.

Cat# iCell-h066

Experimental models: Organisms/strains

C57BL/6JGpt-

F2rl1

em6Cd3832

/Gpt

Gempharmatech

Cat#T006568

Oligonucleotides
Primer of PAR2 (Forward, 5’-
GGCACTCCAGGAAGAAGGCAAAC-

3’; Reverse, 5’-

CAGGGCAGGAATGAAGATGGTCTG-

3’)

Sangon Biotech

N/A

Primer of β-arrestin 2 (Forward, 5’-
TTTTGTTCTTATGCACCCCAAG-

3’; Reverse, 5’-

ATGTCATCATCTGTGGCATAGT-

3’)

Sangon Biotech

N/A

Primer of ERK1(Forward, 5’-
TCTGCTACTTCCTCTACCAGAT-

3’; Reverse, 5’-

CAGGCCGAAATCACAAATCTTA-

3’)

Sangon Biotech

N/A

Primer of ERK2(Forward, 5’-
ATGGTGTGCTCTGCTTATGATA-

3’; Reverse, 5’-

TCTTTCATTTGCTCGATGGTTG-

3’)

Sangon Biotech

N/A

Primer of IL-

17(Forward, 5’-

GAGATATCCCTCTGTGATCTGG-

3’; Reverse, 5’-

GACAGAGTTCATGTGGTAGTCC-

3’)

Sangon Biotech

N/A

Primer of ZO-

1(Forward, 5’-

ACATTGCCGCCAGCCATCTC-

3’; Reverse, 5’-

ATCTATCCACACCATCAGCTTCAGG-

3’)

Sangon Biotech

N/A

Primer of Occludin (Forward, 5’-
AACTTCGCCTGTGGATGACTTC-

3’; Reverse, 5’-

TTTGACCTTCCTGCTCTTCCCTTTG-

3’)

Sangon Biotech

N/A

Primer of Claudin-1 (Forward,

5’-

AGGTACGAATTTGGTCAGGCTCTC-

3’; Reverse, 5’-

GGGACAGGAACAGCAAAGTAGGG-

3’)

Sangon Biotech

N/A

Primer of GAPDH (Forward, 5’-
TGCACCACCAACTGCTTAG-

3’; Reverse, 5’-

AGTAGAGGCAGGGATGATGTTC-

3’)

Sangon Biotech

N/A

siRNA of PAR2 (Sense, 5’-
CCAUGUACCUGAUCUGCUUTT-

3’; Antisense, 5’-

AAGCAGAUCAGGUACAUGGTT-

3’)

GenePharma

N/A

siRNA of β-arrestin 2 (Sense, 5’-
GACCGACUGCUGAAGAAGUTT-

3’; Antisense, 5’-

ACUUCUUCAGCAGUCGGUCTT-

3’)

GenePharma

N/A

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