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The temperature-sensitive receptors TRPV4 and TRPM8 have important roles in the pruritus of rosacea

  • Author Footnotes
    1 These authors contributed equally to this work.
    Xiyuan Zhou
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology, West China Hospital, Sichuan University, Chengdu, China

    Institute of Dermatology and Venereology, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology, Chengdu, China
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  • Author Footnotes
    1 These authors contributed equally to this work.
    Yaoxi Su
    Footnotes
    1 These authors contributed equally to this work.
    Affiliations
    Department of Dermatology, West China Hospital, Sichuan University, Chengdu, China
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  • Shuwei Wu
    Affiliations
    Department of Dermatology, West China Hospital, Sichuan University, Chengdu, China
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  • Hao Wang
    Affiliations
    Department of Dermatology, West China Hospital, Sichuan University, Chengdu, China

    Laboratory of Dermatology, Clinical Institute of Inflammation and Immunology (CIII), Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Sichuan University, Chengdu, China
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  • Ruotian Jiang
    Affiliations
    Laboratory of Anesthesia and Critical Care Medicine, National-Local Joint Engineering Research Center of Translational Medicine of Anesthesiology, West China Hospital, Sichuan University, Chengdu, China
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  • Xian Jiang
    Correspondence
    Correspondence to: Department of Dermatology, West China Hospital, Sichuan University, No.37 Guoxue Alley, Wuhou Dstrict, Chengdu, Sichuan, China.
    Affiliations
    Department of Dermatology, West China Hospital, Sichuan University, Chengdu, China

    Laboratory of Dermatology, Clinical Institute of Inflammation and Immunology (CIII), Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Sichuan University, Chengdu, China
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  • Author Footnotes
    1 These authors contributed equally to this work.
Open AccessPublished:November 30, 2022DOI:https://doi.org/10.1016/j.jdermsci.2022.11.004

      Highlights

      • The subjective symptoms of rosacea are complex and affect patients’ quality of life.
      • Pruritus of rosacea is not purely due to burning, stinging sensations.
      • Temperature changes play an important role in the onset of pruritus.
      • LL-37 intradermally injected on the face of mice could evoke lesions and scratches.
      • The TRPV4 and TRPM8 are related to the pruritus of rosacea.

      Abstract

      Background

      Certain sensations are the secondary phenotypes of rosacea and affect patients’ quality of life. Transient receptor potential (TRP) channels may be involved in its occurrence. However, there is a lack of research independently discussing itch in rosacea.

      Objectives

      Our study aimed to investigate risk factors for pruritus in rosacea patients and to discover the molecular mechanism of pruritus.

      Methods

      A binary logistic regression model was used to identify significant variables affecting pruritus in 782 rosacea patients. The LL-37 was injected intradermally into the face of mice to establish the animal model. qRT–PCR, immunohistochemistry and immunofluorescence were used to analyse the expression differences in pruritus-related molecules in mouse skin and the corresponding trigeminal ganglion (TG) between pruritus and nonpruritus groups.

      Results

      The incidence of pruritus in rosacea was 42.46%, and the incidence of other symptoms increased with pruritus. Temperature effects were prominently related to the itch sensation of rosacea. Intradermal injection of LL-37 not only caused rosacea-like facial lesions but also induced a behavioural pattern indicative of pruritus. Increased expression of the temperature-sensitive receptors TRPV4 and TRPM8 was found in pruritic mouse skin and TG and human skin samples.

      Conclusions

      In rosacea patients, pruritus occurs frequently along with burning, flushing and sensitivity, most likely due to changes in temperature. The temperature-sensitive receptors TRPV4 and TRPM8 are both involved in the mechanism of pruritus in rosacea.

      Abbreviations:

      TRP ((Transient receptor potential)), TRPV4 ((TRP Vanilloid 4)), TRPM8 ((TRP Melastatin 8)), TG ((Trigeminal ganglion)), TRPV ((TRP Vanilloid)), TRPV1 ((TRP Vanilloid 1)), TRPV2 ((TRP Vanilloid 2)), TRPV3 ((TRP Vanilloid 3)), TRPA1 ((TRP Ankyrin 1)), qRT–PCR ((real-time quantitative polymerase chain reaction)), GRP ((Gastrin Releasing Peptide)), NPPB ((Natural polypeptide b))

      Keywords

      1. Introduction

      Rosacea is a chronic cutaneous disorder characterized by transient or persistent erythema, telangiectasia, papules and pustules, primarily affecting the central face. Rosacea is frequently accompanied by discomfort, including burning, stinging and itching [
      • Alexis A.F.
      • Callender V.D.
      • Baldwin H.E.
      • Desai S.R.
      • Rendon M.I.
      • Taylor S.C.
      Global epidemiology and clinical spectrum of rosacea, highlighting skin of color: review and clinical practice experience.
      ]. These sensations are important minor features for the diagnosis of rosacea [
      • van Zuuren E.J.
      • Arents B.W.M.
      • van der Linden M.M.D.
      • Vermeulen S.
      • Fedorowicz Z.
      • Tan J.
      Rosacea: new concepts in classification and treatment.
      ]. According to clinical observations, itch is an unpleasant sensation bothering a certain proportion of patients with rosacea, profoundly impairing the overall quality of life [
      • Oussedik E.
      • Bourcier M.
      • Tan J.
      Psychosocial burden and other impacts of rosacea on patients' quality of life.
      ]. However, the aetiology and pathogenesis of itch in rosacea remain unclear, leaving many sufferers insufficiently treated [
      • Roh Y.S.
      • Choi J.
      • Sutaria N.
      • Kwatra S.G.
      Itch: epidemiology, clinical presentation, and diagnostic workup.
      ]. It is known that the initiation of itch sensation is mediated by the interaction between neuromediators and itch receptors [
      • Choi J.E.
      • Di Nardo A.
      Skin neurogenic inflammation.
      ]. Current evidence suggests that there are two major categories of itch receptors, namely, G protein–coupled receptors and transient receptor potential (TRP) channels [
      • Sutaria N.
      • Adawi W.
      • Goldberg R.
      • Roh Y.S.
      • Choi J.
      • Kwatra S.G.
      Itch: pathogenesis and treatment.
      ]. Interestingly, it was found that TRP channels were closely related to the pathogenesis of rosacea.
      The expression of TRPV channels, including TRPV1, TRPV2, TRPV3 and TRPV4, is generally increased in rosacea skin [
      • Sulk M.
      • Seeliger S.
      • Aubert J.
      • Schwab V.D.
      • Cevikbas F.
      • Rivier M.
      • et al.
      Distribution and expression of non-neuronal transient receptor potential (trpv) ion channels in rosacea.
      ]. Moreover, LL-37 can increase TRPV4 expression directly, which is a necessary condition for the degranulation of mast cells and participates in the inflammation of rosacea [
      • Mascarenhas N.L.
      • Wang Z.
      • Chang Y.L.
      • Di Nardo A.
      Trpv4 mediates mast cell activation in cathelicidin-induced rosacea inflammation.
      ]. The activation of TRPV1 and TRPA1 also causes the release of important mediators of sensory pathologies and neurogenic inflammation, including substance P and calcitonin gene-related peptide, which are also implicated in the pathogenesis of rosacea [
      • Dias M.
      • Newton D.
      • McLeod G.
      • Belch J.J.
      • Khan F.
      Vasoactive properties of calcitonin gene-related peptide in human skin.
      ,
      • Pedersen-Bjergaard U.
      • Nielsen L.B.
      • Jensen K.
      • Edvinsson L.
      • Jansen I.
      • Olesen J.
      Calcitonin gene-related peptide, neurokinin a and substance p: effects on nociception and neurogenic inflammation in human skin and temporal muscle.
      ,
      • Greeno E.W.
      • Mantyh P.
      • Vercellotti G.M.
      • Moldow C.F.
      Functional neurokinin 1 receptors for substance p are expressed by human vascular endothelium.
      ]. TRP channels can be activated by the typical triggering factors of rosacea, including thermal, physical and chemical stimuli [
      • Choi J.E.
      • Di Nardo A.
      Skin neurogenic inflammation.
      ,
      • Gerber P.A.
      • Buhren B.A.
      • Steinhoff M.
      • Homey B.
      Rosacea: the cytokine and chemokine network.
      ,
      • Schwab V.D.
      • Sulk M.
      • Seeliger S.
      • Nowak P.
      • Aubert J.
      • Mess C.
      • et al.
      Neurovascular and neuroimmune aspects in the pathophysiology of rosacea.
      ]. For instance, TRPV1 can be activated by heat, capsaicin or inflammation [
      • Sulk M.
      • Seeliger S.
      • Aubert J.
      • Schwab V.D.
      • Cevikbas F.
      • Rivier M.
      • et al.
      Distribution and expression of non-neuronal transient receptor potential (trpv) ion channels in rosacea.
      ]. TRPV4 can be activated by heat, inflammatory metabolites and osmotic stress, while TRPA1 and TRP melastatin 8 (TRPM8) can be activated by cold and menthol [
      • Sulk M.
      • Seeliger S.
      • Aubert J.
      • Schwab V.D.
      • Cevikbas F.
      • Rivier M.
      • et al.
      Distribution and expression of non-neuronal transient receptor potential (trpv) ion channels in rosacea.
      ,
      • Chuquilin M.
      • Alghalith Y.
      • Fernandez K.H.
      Neurocutaneous disease: cutaneous neuroanatomy and mechanisms of itch and pain.
      ]. Because itch-related TRP channels are also implicated in the pathogenesis of rosacea, these receptors may play a role in the itch sensation of this condition as sensors for certain triggering factors [
      • Gerber P.A.
      • Buhren B.A.
      • Steinhoff M.
      • Homey B.
      Rosacea: the cytokine and chemokine network.
      ,
      • Ahn C.S.
      • Huang W.W.
      Rosacea pathogenesis.
      ]. However, little evidence has been provided to elucidate the potential role of these receptors in itch in rosacea, presumably due to the lack of clinical data and appropriate in vivo models.
      The aetiology and pathogenesis of itch in rosacea patients remain to be elucidated to further alleviate this unpleasant sensation. Therefore, the aim of this study was to (1) identify the potential triggering factors of itch sensation in rosacea patients by a logistic regression model; (2) model facial pruritus in rosacea-like mice by LL-37 injection; (3) investigate the potential role of specific channels and receptors in itch in rosacea by real-time quantitative polymerase chain reaction (qRT–PCR), immunohistochemistry and immunofluorescence; and (4) analyse staining results by semiquantitative analysis. Cathelicidin LL-37 (Cath LL-37) was used to successfully model rosacea-like lesions [
      • Yamasaki K.
      • Di Nardo A.
      • Bardan A.
      • Murakami M.
      • Ohtake T.
      • Coda A.
      • et al.
      Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea.
      ,
      • Muto Y.
      • Wang Z.
      • Vanderberghe M.
      • Two A.
      • Gallo R.L.
      • Di Nardo A.
      Mast cells are key mediators of cathelicidin-initiated skin inflammation in rosacea.
      ] and was involved in pruritus through mast cells [
      • Niyonsaba F.
      • Someya A.
      • Hirata M.
      • Ogawa H.N.
      Evaluation of the effects of peptide antibiotics human beta-defensins-1/-2 and ll-37 on histamine release and prostaglandin d(2) production from mast cells.
      ,
      • Niyonsaba F.
      • Ushio H.
      • Hara M.
      • Yokoi H.
      • Tominaga M.
      • Takamori K.
      • et al.
      Antimicrobial peptides human beta-defensins and cathelicidin ll-37 induce the secretion of a pruritogenic cytokine il-31 by human mast cells.
      ]. Therefore, we injected LL-37 into the facial skin of mice. Finally, we successfully simulated the rosacea-like rash and observed patterns of behaviour indicating pruritus. Then, we found that the expression of TRPV4 and TRPM8 was significantly upregulated in the skin tissues of pruritus mice and rosacea patients compared with those of nonpruritus mice. As critical sensors for heat and cold, respectively, the overexpression of TRPV4 and TRPM8 in rosacea patients with pruritus may explain the involvement of thermal factors in itch sensation suggested by our clinical observations.

      2. Materials and methods

      2.1 Patients and statistics

      A total of 782 patients with a diagnosis of rosacea [
      • Schaller M.
      • Almeida L.M.C.
      • Bewley A.
      • Cribier B.
      • Del Rosso J.
      • Dlova N.C.
      • et al.
      Recommendations for rosacea diagnosis, classification and management: update from the global rosacea consensus 2019 panel.
      ] between October 10th, 2019, and August 31st, 2021, were recruited in our study and divided into two groups based on whether they had rosacea lesion itch. Those patients with other pruritic skin diseases on the face were excluded. Demographic and clinical characteristics, including diet and skin care habits and potential risk factors, were compared between the two groups. Variables with P < 0.1 were included in the binary logistic regression model. Univariate analysis was used to compare the variables, and these significant variables or those considered clinically influential were analysed by multivariate stepwise logistic regression (backwards stepwise logistic regression) to identify significant factors implicated in pruritus in rosacea patients. The statistical analysis was performed using SPSS version 23.0. All the clinical data and specimens were approved by Sichuan University in Chengdu, China (Protocol 2019–248).

      2.2 Animal behavioural studies

      Seven-week-old female BALB/c mice purchased from Hunan Silaike Jingda laboratory Animal Co., Ltd. (Hunan, China) were randomly divided into two groups: the PBS group (n = 18) and the LL-37 group (n = 18). All animal experiments were approved by the Animal Ethics Committee of the Sichuan Academy of Medical Sciences and Sichuan Provincial People’s Hospital (Protocol 2021–246). LL-37 peptide was synthesized by CS Bio Co., Ltd (Shanghai, China) and dissolved in phosphate-buffered saline (PBS). The intradermal injection in the mouse cheek was performed as previously described [
      • Roberson D.P.
      • Gudes S.
      • Sprague J.M.
      • Patoski H.A.
      • Robson V.K.
      • Blasl F.
      • et al.
      Activity-dependent silencing reveals functionally distinct itch-generating sensory neurons.
      ]. For behavioural tests, animals were habituated to separate, clear, plastic containers at least 2 days before observation. Twelve hours after the last injection, their behaviors were separately videoed in the containers for at least 30 min. Behavior and lesion assessments were performed by observers blinded to thegrouping. The hind paws lifted from the floor and scratched the rash, then placed on the floor or in the mouth to count as a bout of scratching [
      • Shimada S.G.
      • LaMotte R.H.
      Behavioral differentiation between itch and pain in mouse.
      ,
      • Oetjen L.K.
      • Mack M.R.
      • Feng J.
      • Whelan T.M.
      • Niu H.
      • Guo C.J.
      • et al.
      Sensory neurons co-opt classical immune signaling pathways to mediate chronic itch.
      ]. Rosacea-like skin lesions were evaluated according to 5 degrees of severity [
      • Li Y.
      • Xie H.
      • Deng Z.
      • Wang B.
      • Tang Y.
      • Zhao Z.
      • et al.
      Tranexamic acid ameliorates rosacea symptoms through regulating immune response and angiogenesis.
      ]. The detailed process is shown in a flowchart (Figure A). The pruritus (LL-37) group was defined by significantly more than eight itching bouts, which was the maximum in the PBS group. Statistics for this part of the study were performed by GraphPad Prism 7.04 (GraphPad Software Inc., CA, USA).

      2.3 Quantitative RT–PCR

      Total RNA was extracted from skin or TG samples using a FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme Cat., Nanjing, China). cDNA was synthesized from 1000 ng of total RNA by a Prime ScriptTM RT Reagent Kit with gDNA Eraser (Perfect Real Time) purchased from Takara Biomedical Technology Co., Ltd. (Beijing, China). Each skin cDNA sample was amplified for 15 genes related to itching and TNF-α and IL-1β related to rosacea inflammation. TG cDNA samples were amplified for genes related to itching only.
      Reactions were performed in a 20 µl reaction volume using TB Green Premix Ex TaqⅡ (Tli RNaseH Plus) (Takara Biomedical Technology Co., Ltd, Beijing, China) and ABI QuantStudio 6 Flex (Thermo Fisher Scientific, MA, USA). The real-time PCR conditions were 95 °C for 30 s followed by 40 cycles at 95 °C for 5 s and 60 °C for 34 s. The mRNA levels of all genes were normalized to GAPDH and calculated using the 2-△△CT method. The comparisons were performed using a t-test by SPSS version 23.0. and P value < 0.05 was considered statistically significant. The primer sequences (5′ to 3′) involved are shown in Supplementary Table B.

      2.4 Immunohistochemistry

      Tissues were treated with EDTA pH 9.0 (ZSGB Bio, Beijing, China) for antigen retrieval and then incubated with primary antibodies against TRPV4 (catalogue No. 20987–1-AP, dilution 1:200, Proteintech) and TRPM8 (catalogue No. 12813–1-AP, dilution 1:200, Proteintech) overnight at 4 °C. Then, tissues were incubated with goat anti-rabbit secondary antibody (catalogue No. ZB-2301, dilution 1:250, ZSGB Bio) for 40 min at room temperature. Images were captured using NIS-Elements AR (Nikon, Japan) and analysed by ImageJ analysis software (NIH, USA) with IHC Profiler Plugin. Five nonoverlapping, random fields for the skin (×400 total magnification) and 3 for the TG of each mouse were examined. The region of interest (ROI) of skin tissue was in the epidermis.

      2.5 Immunofluorescence

      We used anti-Mast Cell Tryptase staining to detect mast cell activation. Tissues were treated with citric acid (ZSGB Bio, Beijing, China) for antigen retrieval and then incubated with primary antibody against Tryptase of Mast Cell (catalog No. ab2378, dilution 1:2000, Abcam) overnight at 4 °C. Then, tissues were incubated with Alexa Fluor® 488 AffiniPure goat anti-mouse IgG (H+L) secondary antibody (catalog No. 111–485–003, dilution 1:200, Jackson) for 40 min at room temperature. TRPV4 and TRPM8 were double stained. After antigen repair, tissues were incubated with primary antibody against TRPV4 (catalog No. 20987–1-AP, dilution 1:200, Proteintech) overnight and washed four times after incubation with Alexa Fluor® 488 AffiniPure goat anti-rabbit IgG (H+L) secondary antibody (catalog No. 111–545–003, dilution 1:200, Jackson) for 40 min at room temperature. Then, tissues were incubated with TRPM8 primary antibody (catalog No. 12813–1-AP, dilution 1:200, Proteintech) overnight at 4 °C again. Finally, the secondary antibody CyTM3 AffiniPure Goat Anti-Rabbit IgG (H+L) (catalog No. 111–165–003, dilution 1:200, Jackson) was incubated with the tissue for 90 min. Images were captured using a Leica s-STELLARIS (Leica, Germany) and analyzed by ImageJ analysis software (NIH, USA).

      3. Results

      3.1 Population characteristics and pruritus-related risk factors

      Of the 782 patients, 332 (42.46%) were assigned to the pruritus group, and 450 (57.54%) were assigned to the nonpruritus group according to their itching condition. The statistically significant variables are summarized in Table A. The results of logistic regression analysis assessing 6 potential risk factors are presented in Table 1 and Fig. 1a. Two of these 6 factors are related to temperature, including aggravation by both cold and heat (hot and cold biphase) and the seasons and their corresponding temperature change. The odds ratio (OR) values were 1.518 [95% confidence interval (CI) 1.019–2.263, P = 0.040] and 1.449 [95% confidence interval (CI) 1.023–2.054, P = 0.037], respectively. Statistical analysis was performed on concomitant clinical symptoms and signs (Fig. 1b). Nine of ten features in the pruritus group occurred significantly more often than in the nonpruritus group, with burning, flushing and sensitivity occurring in 72.89% (P < 0.001), 69.88% (P < 0.05) and 68.98% (P < 0.01) of patients in the pruritus group, respectively.
      Table 1Univariate and Multivariate logistic regression assessing risk factors for pruritus in rosacea.
      VariableUnivariateMultivariate
      OR(95% CI)p valueOR(95% CI)p value
      Gender
       Female2.078(1.327–3.254)0.001
      P﹤0.01,
      1.502(0.902–2.499)0.118
       Maleref
      Smoke1.338(0.990–1.808)0.0581.395(1.015–1.917)0.040
      P﹤0.05,
      Classification————————
       Phymatous0.312(0.167–0.583)<0.001
      P﹤0.001
      0.435(0.215–0.878)0.020
      P﹤0.05,
      Comorbidities (pruritic dermatosis)2.335(1.443–3.805)0.001
      P﹤0.01,
      2.187(1.309–3.656)0.003
      P﹤0.01,
      Comorbidities (systemic diseases)————————
       Gastrointestinal1.799(1.097–2.950)0.020
      P﹤0.05,
      1.441(0.848–2.447)0.177
       Neurologic1.927(1.001–3.332)0.049
      P﹤0.05,
      1.289(0.678–2.453)0.439
       Respiratory3.107(0.949–10.176)0.0612.639(0.783–8.891)0.117
      Aggravating factors————————
       Dry1.639(1.139–2.360)0.008
      P﹤0.01,
      1.163(0.788–1.718)0.447
       Stress1.650(1.222–2.227)0.001
      P﹤0.01,
      1.397(1.013–1.926)0.041
      P﹤0.05,
       Ultraviolet1.340(1.008–1.782)0.044
      P﹤0.05,
      1.081(0.799–1.461)0.614
      Hot and cold biphasic1.841(1.258–2.694)0.002
      P﹤0.01,
      1.518(1.019–2.263)0.040
      P﹤0.05,
      Seasons of recurrence or exacerbation

      Spring or/and autumn (temperature change)
      ——

      1.674(1.202–2.332)
      ——

      0.002
      P﹤0.01,
      ——

      1.449(1.023–2.054)
      ——

      0.037
      P﹤0.05,
      * P﹤0.05,
      ** P﹤0.01,
      *** P﹤0.001
      Fig. 1
      Fig. 1Six factors are implicated in itch sensation of rosacea patients and other accompanying symptoms. a. Forest plot showing 6 factors related to itch of rosacea, 2 of which are related to temperature changes. (* P﹤0.05, ** P﹤0.01, *** P﹤0.001 versus nonpruritus, Chi Square test). b. Other clinical features accompanied by itch (%= n/332), 9 of 10 rosacea symptoms were significantly increased in the pruritus group.

      3.2 Construction of the LL-37-induced pruritus mouse model and erythema assessment

      Inflammatory lesions were elicited by intradermal injections of LL-37 in cheek skin, and behavioural responses to pruritus were subsequently observed as the number of bouts (Fig. 2). The severity of the erythema of skin lesions was evaluated and compared between the PBS group and the LL-37 group, and significantly higher scores were found in the LL-37 group (P < 0.0001. Fig. 2b, Figure B). The frequency of bouts was generally higher in the LL-37 group (P < 0.01. Fig. 2c). Eight of eighteen (44.44%) mice injected with LL-37 were found to have pruritus according to the frequency of bouts, which was not significantly different from that in pruritus patients in our clinical data (42.46%, P = 0.866). However, there was no correlation between erythema severity scores and the number of bouts (P = 0.918. Fig. 2d).
      Fig. 2
      Fig. 2More scratching bouts of mice were observed in the LL-37 group, and upregulated mRNA expression of TRPV4 and TRPM8 is shown in both skin tissues and trigeminal ganglia of pruritus mice. a. Representative histopathological H&E staining of skin lesions in the LL-37 group:thickened epidermis and increased inflammatory cell infiltration compared with the PBS group. b-c. Both erythema scores (b) and scratching bouts (c) of the LL-37 group were greater than those in the PBS group (**** P<0.0001, ** P<0.01). d. No significant correlation between the number of scratching bouts and erythema severity scores was found (P = 0.918). e. Five out of seventeen genes with differential expression in the skin tissues of pruritus mice(LL-37, n = 4), nonpruritus mice(LL-37, n = 4)and control mice(PBS, n = 4)by real-time quantitative polymerase chain reaction (qRT–PCR) (** P<0.01, # P<0.05 and the expression in the nonpruritic group was higher). Interestingly, the expression of proinflammatory cytokines (TNF-α and IL-1β) was not upregulated in the skin tissues of pruritus mice compared with those of the nonpruritus. f. Differential expression of the genes of interest in the corresponding Trigeminal ganglion (TG) of three groups (n = 4 in each group) by qRT-PCR, (* P<0.05).

      3.3 Upregulated expression of TRPV4 and TRPM8 in the skin and trigeminal ganglia of pruritus mice

      The expression levels of the 17 genes in skin biopsy samples were detected by qRT–PCR. The mRNA levels of TRPV4 (P < 0.01), TRPM8 (P < 0.01) and gastrin-releasing peptide (GRP) (P < 0.01) in pruritus mice were significantly increased compared with those in nonpruritus mice, as shown in Fig. 2e. Similar results were presented in trigeminal ganglia (TG) (P < 0.05. Fig. 2f). TNF-α and IL-1β, crucial cytokines implicated in the inflammation of rosacea, did not increase in the skin tissue of the pruritus group.

      3.4 Increased expression of Mast Cell Tryptase, TRPV4 and TRPM8 were found in pruritus mice

      The expression of Mast Cell Tryptase in the pruritus group was higher than that in the non-pruritus group and PBS group in immunofluorescence (Figure C). The distribution of TRPV4 and TRPM8 in the skin and TG of mice was observed by immunohistochemistry and immunofluorescence, as shown separately in Fig. 3a and Fig. 4a. The immunohistochemical results of the samples above both illustrated an increase in TRPV4 and TPRM8 colocalized to the membrane of epidermal cells. Epidermal TRPV4 and TRPM8 were rated semiquantitatively, which showed increased staining in pruritus skin versus nonpruritus skin (P < 0.05. Fig. 3b-d). Similar results were observed in TG, as shown in Fig. 4b-d. In conclusion, increased immunoreactivity of TRPV4 and TRPM8 was found in the skin and TG of mice in the pruritus group. However, double immunofluorescence revealed that the colocalization of TRPV4 and TRPM8 was not significantly observed in TG (Pearson's R value < 0.8, Fig. 4e-h).
      Fig. 3
      Fig. 3The expression of TRPV4 and TRPM8 is upregulated in the skin tissues of pruritus mice versus nonpruritus mice. a. Localization and expression of TRPV4 and TRPM8 in the skin tissues from 3 groups by immunohistochemistry and immunofluorescence. Immunofluorescence: TRPV4 (488 nm, green), TRPM8 (550 nm, red) and nuclei DAPI (blue). Scale bares: 50 µm (×400 magnification). b-c. Quantification of TRPV4 (b) and TRPM8 (c) in the epidermis by immunohistochemistry (n = 3). The percentage contibution of positive was evaluated by Image J software, (**** P<0.0001, ** P<0.01). d. Quantification of TRPV4 and TRPM8 in the epidermis by immunofluorescence (n = 3). The integrated density (IntDen) was compared between 3 groups, (*** P<0.001).
      Fig. 4
      Fig. 4The expression of TRPV4 and TRPM8 is upregulated in Trigeminal ganglia (TG) in the corresponding lesions of pruritus mice versus nonpruritus mice. a. Localization and expression of TRPV4 and TRPM8 in TG of three groups by immunohistochemistry and immunofluorescence. Scale bares: 50 µm (×400 magnification) for immunohistochemistry, 10 µm (×400 magnification) for immunofluorescence. b-c. Quantification of TRPV4(b) and TRPM8(c) in the TG tissue by immunohistochemistry (n = 3). The percentage contibution of positive was evaluated by Image J software, (**** P<0.0001, * P<0.05). d. Quantification of TRPV4 and TRPM8 in the corresponding TG samples by immunofluorescence (n = 3). The integrated density (IntDen) was compared between 3 groups (**** P<0.0001, *** P<0.001). e. Colocalization of TRPV4 and TRPM8 in TG of pruritus mice by immunofluorescence. Orange circles show the areas where TRPV4 and TRPM8 are colocalized. f-h: Colocalization expression analysis of TRPV4 and TRPM8 by immunofluorescence in image TG1 (f), TG2 (g) and TG3(h) respectively from 3 pruritus specimens. The Pearson’s R value were 0.70, 0.60 and 0.71 respectively.

      3.5 Upregulation of TRPV4 and TRPM8 expression in the skin lesions of rosacea patients with pruritus

      A set of immunofluorescence stained skin biopsy samples from pruritus and nonpruritus rosacea patients, as well as healthy controls, is shown in Fig. 5a. Immunofluorescent staining of human skin successfully labelled TRPV4 and TRPM8 in the epidermis. It showed that the fluorescence of TRPV4 and TRPM8 antibodies was obviously enhanced in the epidermal cell membrane of pruritus patients, compared with nonpruritus patients and the healthy population. An increased intensity of epidermal staining was shown by semiquantitative analysis (P < 0.01, Fig. 5b).
      Fig. 5
      Fig. 5The expression of TRPV4 and TRPM8 is significantly upregulated in skin lesions of pruritus rosacea patients versus nonpruritus patients. a. Localization and expression of TRPV4 and TRPM8 in skin lesions of pruritus patients, nonpruritus rosacea patients and healthy controls by immunofluorescence. Scale bares: 50 µm (×400 magnification). b. Quantification of TRPV4 and TRPM8 in the epidermis of human skin tissues by immunofluorescence (n = 3). The integrated density (IntDen) was evaluated by Image J software (** P<0.01, * P<0.05).

      4. Discussion

      Pruritus has been reported in 36.51% of patients with rosacea [
      • Huet F.
      • Taieb C.
      • Corgibet F.
      • Brenaut E.
      • Richard M.A.
      • Misery L.
      Pruritus, pain, and depression associated with the most common skin diseases: Data from the french study "objectifs peau".
      ]. According to our clinical observations, 42.46% of rosacea patients complained of pruritus, which was consistent with our animal experiment results that 44.44% of mice displayed scratching behaviour due to pruritus elicited by LL-37. Through the comparison of the two clinical groups with and without pruritus, our results supported the previous findings that certain factors can elicit the occurrence of itch, such as dryness [
      • Moniaga C.S.
      • Tominaga M.
      • Takamori K.
      Mechanisms and management of itch in dry skin.
      ], stress [
      • Sanders K.M.
      • Akiyama T.
      The vicious cycle of itch and anxiety.
      ], ultraviolet radiation and factors related to hot and cold temperatures. The simultaneous effects of cold and heat can aggravate itching, which often occurs in spring and autumn. However, the severity of the disease was not among these factors, which was also confirmed later by the animal model. The classical inflammatory factor TNF-α did not increase in the pruritus group, while IL-1β was even higher in the non-pruritus group. It seems that pruritus is not associated with inflammatory conditions or the erythema severity of rosacea, as mentioned before [
      • Choi J.E.
      • Di Nardo A.
      Skin neurogenic inflammation.
      ,
      • Erickson S.
      • Heul A.V.
      • Kim B.S.
      New and emerging treatments for inflammatory itch.
      ]. Nevertheless, 2 of the 6 factors are related to temperature, indicating the importance of temperature changes, cold and heat in the pathogenesis of pruritus in rosacea.
      It was previously known that itching was not only a symptom of a disease, but the result of interaction between the skin, ganglia, and brain regarding the immune and nervous systems [
      • Sutaria N.
      • Adawi W.
      • Goldberg R.
      • Roh Y.S.
      • Choi J.
      • Kwatra S.G.
      Itch: pathogenesis and treatment.
      ]. Inflammatory factors produced by other inflammatory skin diseases can also cause itching due to immune disorders [
      • Trier A.M.
      • Kim B.S.
      Cytokine modulation of atopic itch.
      ,
      • Garcovich S.
      • Maurelli M.
      • Gisondi P.
      • Peris K.
      • Yosipovitch G.
      • Girolomoni G.
      Pruritus as a distinctive feature of type 2 inflammation.
      ]. In our study, other pruritic skin disorders were also found to be a risk factor of itching. Additionally, both our research and previous studies have shown that itching is not always an independent feeling, which was embodied in the fact that patients with pruritus are often accompanied by burning, prickling, and stinging [
      • Cevikbas F.
      • Lerner E.
      Physiology and pathophysiology of itch.
      ]. The complexity of rosacea pruritus may be an important reason for the lack of previous research.
      Another reason for the rarity of research on subjective symptoms of rosacea is the lack of animal models. LL-37 is an antibacterial peptide produced by epithelial tissue against damage, ultraviolet rays, and pathogenic microorganisms [
      • Yoon S.H.
      • Hwang I.
      • Lee E.
      • Cho H.J.
      • Ryu J.H.
      • Kim T.G.
      • et al.
      Antimicrobial peptide ll-37 drives rosacea-like skin inflammation in an nlrp3-dependent manner.
      ]. Intradermal injection of LL-37 induced a rosacea-like rash in mice but was not found to induce scratching behavior [
      • Reddy V.B.
      • Graham T.A.
      • Azimi E.
      • Lerner E.A.
      A single amino acid in mrgprx2 necessary for binding and activation by pruritogens.
      ]. LL-37 may also relieve itching in atopic dermatitis by increasing nerve repulsion factors [
      • Umehara Y.
      • Kamata Y.
      • Tominaga M.
      • Niyonsaba F.
      • Ogawa H.
      • Takamori K.
      Cathelicidin ll-37 induces semaphorin 3a expression in human epidermal keratinocytes: implications for possible application to pruritus.
      ]. Although LL-37 has no direct pruritic effect, it has been reported to induce mast cells to release histamine and the itch-derived cytokine IL-31 [
      • Niyonsaba F.
      • Someya A.
      • Hirata M.
      • Ogawa H.N.
      Evaluation of the effects of peptide antibiotics human beta-defensins-1/-2 and ll-37 on histamine release and prostaglandin d(2) production from mast cells.
      ,
      • Niyonsaba F.
      • Ushio H.
      • Hara M.
      • Yokoi H.
      • Tominaga M.
      • Takamori K.
      • et al.
      Antimicrobial peptides human beta-defensins and cathelicidin ll-37 induce the secretion of a pruritogenic cytokine il-31 by human mast cells.
      ]. In our study, we found that LL-37 did not cause an increase in IL-31 in the mouse skin. However, the expression of Trpytase, a marker of mast cell activation [
      • Atiakshin D.
      • Buchwalow I.
      • Samoilova V.
      • Tiemann M.
      Tryptase as a polyfunctional component of mast cells.
      ], increased in the pruritus group. This suggests that LL-37 may cause itching behavior through mast cell activation. However, not all mice had pruritus after LL-37 injection (only 44%), which was consistent with mast cell activation. The relationship between LL-37 and mast cell activation remains to be further studied.
      In addition, two neuropeptides related to pruritus were also found to be different between groups in this study. As an itch specific neurotransmitter and vasodilator, GRP may participate in flushing, telangiectasia, stinging and other relative sensations associated with pruritus of rosacea [
      • Barry D.M.
      • Liu X.T.
      • Liu B.
      • Liu X.Y.
      • Gao F.
      • Zeng X.
      • et al.
      Exploration of sensory and spinal neurons expressing gastrin-releasing peptide in itch and pain related behaviors.
      ]. Natural polypeptide b (NPPB) is expressed on a subset of TRPV1 neurons, and is also coexpressed with neuropeptide Y Y2 receptor in neurons to participate in the itch induced by IL-31 [
      • Mishra S.K.
      • Hoon M.A.
      The cells and circuitry for itch responses in mice.
      ,
      • Ma H.
      • Gao T.
      • Jakobsson J.E.T.
      • Weman H.M.
      • Xu B.
      • Larhammar D.
      • et al.
      The neuropeptide y y2 receptor is coexpressed with nppb in primary afferent neurons and y2 activation reduces histaminergic and il-31-induced itch.
      ]. Although NPPB is closely related to pruritus, it may not be involved in the conscious symptoms of rosacea. Because it and its related TRPV1, IL-31 was not increased in the pruritus group in this study. However, its increase in the non-pruritus group may require further verification of the sample size.
      Previous work has shown that TRPV4 and TRPM8 can be activated by thermal stimuli [
      • Shibasaki K.
      Trpv4 activation by thermal and mechanical stimuli in disease progression.
      ,
      • Voets T W.U.D.G.
      • Janssens A.
      • Flockerzi V.
      • Nilius B.
      The principle of temperature-dependent gating in cold- and heat-sensitive trp channels.
      ], where TRPV4 is a heat-sensitive channel and TRPM8 is a cold-sensitive channel. In addition, these TRP channels are also have regulatory functions in itch and pain [
      • Moore C G.R.
      • Jordt S.E.
      • Chen Y.
      • Liedtke W.B.
      Regulation of pain and itch by trp channels.
      ]. The enhanced expression levels of TRPV4 and TRPM8 in the skin and TG in pruritus mice suggest that temperature has a potential effect on pruritus through TRPV4 and TRPM8 channels. Furthermore, we observed high expression of TRPV4 and TRPM8 in keratinocytes by immunohistochemistry and fluorescence. Previous study has demonstrated that TRPV4 and TRPM8 can be expressed on various cells including nerve cells, keratinocytes, and immune cells [
      • Moore C.
      • Gupta R.
      • Jordt S.E.
      • Chen Y.
      • Liedtke W.B.
      Regulation of pain and itch by trp channels.
      ,
      • Bidaux G.
      • Borowiec A.S.
      • Gordienko D.
      • Beck B.
      • Shapovalov G.G.
      • Lemonnier L.
      • et al.
      Epidermal trpm8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a cold-dependent manner.
      ]. Not only can TRP on nerve cells be involved in itch, but TRP on keratinocytes can also be directly involved. Activation of TRPV4 causes calcium influx into keratinocytes and upregulates the phosphorylation of mitogen-activated protein kinasein cells. TRPV4 keratinocyte-specific inducible knockout mice have significantly reduced acute histaminergic itch [
      • Chen Y.
      • Fang Q.
      • Wang Z.
      • Zhang J.Y.
      • MacLeod A.S.
      • Hall R.P.
      • et al.
      Transient receptor potential vanilloid 4 ion channel functions as a pruriceptor in epidermal keratinocytes to evoke histaminergic itch.
      ]. In addition, dermal macrophages also express TRPV4, which can co-regulate chronic pruritus with keratinocytes [
      • Luo J.
      • Feng J.
      • Yu G.
      • Yang P.
      • Mack M.R.
      • Du J.
      • et al.
      Transient receptor potential vanilloid 4-expressing macrophages and keratinocytes contribute differentially to allergic and nonallergic chronic itch.
      ]. Activation of TRPV4 induces itching, while activation of TRPM8 mainly induces cold pain [
      • Moore C.
      • Gupta R.
      • Jordt S.E.
      • Chen Y.
      • Liedtke W.B.
      Regulation of pain and itch by trp channels.
      ]. Meanwhile TRPM8 may be responsible for the stinging and burning sensations that accompany the cold pain, increasing the irritation caused by the cold [
      • Selescu T.
      • Ciobanu A.C.
      • Dobre C.
      • Reid G.
      • Babes A.
      Camphor activates and sensitizes transient receptor potential melastatin 8 (trpm8) to cooling and icilin.
      ,
      • Belmonte J.
      • Alfaro S.
      • Berenguer T.B.
      • Costa J.M.
      • Cuquerella V.
      • Acosta M.C.
      • et al.
      Effects of the trpm8 agonist menthol on corneal sensitivity and tear secretion.
      ]. We speculate that the increased expression of TRPM8 in skin and ganglia may be the cause of itching accompanied by stinging and burning in rosacea patients. Increased co-expression of TRPV4 and TRPM8 was associated with the appearance of these subjective symptoms and was also associated with the hot and cold biphasic temperature change.
      The interaction between LL-37 and TRP channels on various cells has been previously reported. In rosacea, LL-37 induces inflammation by activating TRPV4 in mast cells, followed by cell degranulation [
      • Dias M.
      • Newton D.
      • McLeod G.
      • Belch J.J.
      • Khan F.
      Vasoactive properties of calcitonin gene-related peptide in human skin.
      ]. LL-37 can activate TRPV2 and TRPC channels [
      • Tsvilovskyy V.
      • Solis-Lopez A.
      • Almering J.
      • Richter C.
      • Birnbaumer L.
      • Dietrich A.
      • et al.
      Analysis of mrgprb2 receptor-evoked ca (2+) signaling in bone marrow derived (bmmc) and peritoneal (pmc) mast cells of trpc-deficient mice.
      ,
      • Gambade A.
      • Zreika S.
      • Guéguinou M.
      • Chourpa I.
      • Fromont G.
      • Bouchet A.
      • et al.
      Activation of trpv2 and bkca channels by the ll-37 enantiomers stimulates calcium entry and migration of cancer cells.
      ], and TRP channel activation can also affect the production of LL-37 [
      • Majhi R.K.
      • Mohanty S.
      • Kamolvit W.
      • White J.K.
      • Scheffschick A.
      • Brauner H.
      • et al.
      Metformin strengthens uroepithelial immunity against e. Coli infection.
      ]. Although there is no direct correlation with LL-37 for TRPM8, it may play a role in the ocular symptoms of rosacea [
      • Belmonte J.
      • Alfaro S.
      • Berenguer T.B.
      • Costa J.M.
      • Cuquerella V.
      • Acosta M.C.
      • et al.
      Effects of the trpm8 agonist menthol on corneal sensitivity and tear secretion.
      ]. Whether LL-37 can also induce activation of TRPV4 and TRPM8 channels on other immune cells or keratinocytes remains to be investigated.
      In conclusion, on the basis of our clinical data, we confirmed that pruritus in patients with rosacea is often accompanied by complex subjective symptoms. And we suspected that the occurrence of pruritus was related to hot and cold temperatures. LL-37 can cause rosacea-like rash and scratching behavior in mice by multiple intradermal injections at low concentrations. Eventually, we revealed a potential effect of TRPV4 and TRPM8 on rosacea pruritus to some degree. TRPV4 may be responsible for thermal factors and itch, whereas TRPM8 may be responsible for cold stimulation and other complex sensations that accompany itching. However, we also need to distinguish this itch from other types of itch due to other pruritic skin diseases, psychological factors and so on. And the relationship between LL-37 and TRPV4, TRPM8 also needs to be further explored. After all, these efforts are still in their early stages due to the complexity of rosacea itching. We believe novel specific TRPV4 or TRPM8 modulatory chemicals are on the way to offer more options for rosacea patients to relieve their symptoms.

      Funding

      This work was supported by Scientific Research Project on Cadre Health Care in Sichuan Province (2020-229); and supported by Chengdu Technological Innovation Research and Development Project (2021-YF05-000579-SN); and supported by Clinical Research Innovation Project, West China Hospital, Sichuan University (19HXCX010); and supported by National Natural Science Foundation of China (82073473).

      CRediT authorship contribution statement

      Xiyuan Zhou: Methodology, Formal analysis, Investigation,Visualization, Yaoxi Su: Writing – original draft. Shuwei Wu: Writing – review & editing, Hao Wang: Resources, Ruotian Jiang: Methodology, Validation, Xian Jiang: Conceptualization, Project administration, Funding acquisition.

      Conflicts of interest

      The authors have no conflict of interest to declare.

      Acknowledgements

      We would like to thank our patient questionnaire team for their help in collecting and managing clinical data.

      Appendix A. Supplementary material

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