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Mechanisms of itch in stasis dermatitis: Significant role of IL-31 from macrophages

Takashi Hashimoto, MD, PhD, Christina Dorothy Kursewicz, BS, Rachel Alison
Fayne, BA, Sonali Nanda, MS, Serena Maya Shah, BS candidate, Leigh Nattkemper,
PhD, Hiroo Yokozeki, MD, PhD, Gil Yosipovitch, MD
PII: S0022-202X(19)33304-4
DOI: https://doi.org/10.1016/j.jid.2019.09.012
Reference: JID 2165

To appear in: The Journal of Investigative Dermatology

Received Date: 29 June 2019

Revised Date: 24 September 2019
Accepted Date: 30 September 2019

Please cite this article as: Hashimoto T, Kursewicz CD, Fayne RA, Nanda S, Shah SM, Nattkemper
L, Yokozeki H, Yosipovitch G, Mechanisms of itch in stasis dermatitis: Significant role of IL-31
from macrophages, The Journal of Investigative Dermatology (2019), doi: https://doi.org/10.1016/
j.jid.2019.09.012.

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TITLE: Mechanisms of itch in stasis dermatitis: Significant role of IL-31 from macrophages

AUTHORS: Takashi Hashimoto, MD, PhD1),2)*, Christina Dorothy Kursewicz, BS1), Rachel

Alison Fayne, BA1), Sonali Nanda, MS1), Serena Maya Shah, BS candidate1), Leigh Nattkemper,

PhD1), Hiroo Yokozeki, MD, PhD2), Gil Yosipovitch, MD1)*.

1) Miami Itch Center, Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery,

Miller School of Medicine, University of Miami, Miami, FL, US

2) Department of Dermatology, Graduate School of Medical and Dental Sciences, Tokyo Medical

and Dental University, Tokyo, Japan

* Co-corresponding authors

ORCIDs:

Takashi Hashimoto, 0000-0001-6779-5598

Christina Dorothy Kursewicz, 0000-0001-5135-6865

Rachel Alison Fayne, 0000-0002-9350-9812

Sonali Nanda, 0000-0001-5501-8193

Serena Maya Shah, 0000-0002-0981-7792

Leigh Nattkemper, 0000-0003-0895-7985

Hiroo Yokozeki, 0000-0002-5773-9485

Gil Yosipovitch, 0000-0001-6303-1822

CORRESPONDING AUTHORS:

Gil Yosipovitch, MD

1600 NW 10th Ave, RMSB 2067B, Miami, FL 33136

Tel: 305-213-5824

Fax: 305-243-6191

1
 Email: gyosipovitch@med.miami.edu

Takashi Hashimoto, MD, PhD

1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan

Tel: 81-3-5903-5286

Fax: 81-3-5803-5289

Email: hashderm@tmd.ac.jp

SHORT TITLE: Macrophage-derived IL-31 in itchy stasis dermatitis

ABBREVIATIONS: AD (atopic dermatitis), Arg-1 (arginase-1), CCR4 (C-C chemokine

receptor type 4), CXCR3 (C-X-C chemokine receptor type 3), IL (interleukin), MOMA-2

(monocyte/macrophage), NK1R (neurokinin-1 receptor), OSMRβ (oncostatin M receptor β),

PAR-2 (protease-activated receptor 2), pM (peritoneal macrophages), RBC (red blood cell), SD

(stasis dermatitis), SP (Substance P), Th (T helper), TRPA1 (transient receptor potential

ankyrin-1), TRPV1 (transient receptor potential vanilloid-1), TSLP (thymic stromal

lymphopoietin).

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ABSTRACT

Stasis dermatitis (SD) is a common disease in the elderly population, with pruritus being one of

the bothersome symptoms. However, there are few therapeutic modalities available for

SD-associated itch because little is known about its pathophysiological mechanism. Therefore,

we sought to investigate the mediators of itch in SD using an immunofluorescence study on

patient lesions focusing on IL-31. Ex vivo stimulation studies using murine peritoneal

macrophages were also used to elucidate the pathological mechanisms of IL-31 generation. In SD

lesions, dermal infiltrating IL-31(+) cells were increased in number compared to healthy controls,

and the majority of IL-31(+) cells were CD68(+) macrophages. The presence of itch in SD was

significantly associated with the amount of CD68(+)/IL-31(+) macrophages and

CD68(+)/CD163(+) M2 macrophages. The number of CD68(+)/IL-31(+) macrophages was

correlated with the number of dermal CCR4(+) Th2 cells, IL-17(+) cells, basophils, substance

P(+) cells, and dermal deposition of periostin and hemosiderin. Furthermore, murine peritoneal

macrophages expressed an M2 marker arginase-1 and generated IL-31 when stimulated with a

combination of substance P, periostin, and red blood cell lysate (representing hemosiderin). IL-31

from macrophages may play a role in itch in SD.

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INTRODUCTION

Stasis dermatitis (SD) is a common disease that predominantly affects the lower legs of

elderly patients, with a prevalence of 6.2% among those older than 65 years (Yalçin et al. 2006).

The main causes of SD are chronic venous insufficiency and venous hypertension. Venous

hypertension promotes cellular accumulation of inflammatory cells (e.g., T cells and

macrophages) and extravasation of red blood cells (RBCs) in the affected skin (Saharay et al.

1997; Thomas et al. 1988). Extravasation and disruption of RBCs are followed by decomposition

of hemoglobin, which results in excessive tissue iron stored as hemosiderin (Caggiati et al. 2010).

Hemoglobin and hemosiderin can further induce monocyte/macrophage-recruitment through

hemoglobin scavenger receptor CD163 (Rubio-Navarro et al. 2015). Accumulating macrophages

and other cells promote inflammation and induce the abnormal histological features of SD (e.g.,

epidermal spongiotic changes, papillary structure alternation, and capillary proliferation) partially

through the secretion of proteolytic enzymes (Sundaresan et al. 2017; Wenk et al. 2001).

One of the bothersome symptoms reported in SD is pruritus. In an elderly population,

the dermatosis that was most commonly reported to cause the complaint of itch was SD

(Valdes-Rodriguez et al. 2015). Itch in SD not only impairs patients’ quality of life, but also

induces scratching, which aggravates wounds and increases the risk of skin infection.

Unfortunately, there are few therapeutic modalities available for itch because little is known

about the pathophysiological mechanisms of SD-associated itch.

The phenomenon of itch is divided into two subgroups: histaminergic and

non-histaminergic (Yosipovitch et al. 2018). Itch in SD appears to be non-histaminergic, as

antihistamines do not necessarily improve SD-associated itch. Non-histaminergic itch involves

4
various itch mediators including cytokines/chemokines (e.g., interleukin [IL]-31), amines,

proteases and their associated receptors (e.g., protease-activated receptor-2 [PAR-2]),

neuropeptides and receptors (e.g., substance P [SP] and its receptor neurokinin-1 receptor

[NK1R]), ion channels (e.g., transient receptor potential ankyin-1 [TRPA1] and vanilloid-1

[TRPV1]), and immune cells (e.g., T cells, mast cells, eosinophils, and basophils).

IL-31, a T helper (Th)2-related pruritogenic cytokine, has recently gained attention as a

potential therapeutic target for inflammatory skin conditions with itch (Furue et al. 2018). IL-31

is involved in itch in various diseases e.g. atopic dermatitis (AD) (Nattkemper et al. 2018;

Sonkoly et al. 2006), prurigo nodularis (Sonkoly et al. 2006), psoriasis (Nattkemper et al. 2018),

and cutaneous T-cell lymphoma (Nattkemper et al. 2016). IL-31 exerts its function through its

receptor complex, comprising IL-31RA and oncostatin M receptor β (OSMRβ) (Sonkoly et al.

2006). Blocking IL-31RA has been shown to improve itch in AD (Kabashima et al. 2018).

However, the involvement of IL-31 and its receptor complex in SD-associated itch is unknown.

Thus, we sought to investigate the pathophysiological mechanisms of SD-associated itch

focusing on IL-31 and other major itch mediators through an immunofluorescence study of

patient lesions. In addition, we sought to elucidate the pathological mechanisms of IL-31

generation through ex vivo stimulation studies using murine peritoneal macrophages.

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RESULTS

CD68(+)/CD163(+) macrophages express IL-31 and correlate with the presence of itch in

SD

In SD lesions, the number of dermal infiltrating IL-31(+) cells was significantly

increased compared to healthy subjects (t-test, P = 0.001), while epidermal expression was not

(t-test, P = 0.80). In addition, SD with severe itch, which was defined as level 4 itch using the

Likert scale from zero (no itch) to 4 (severe itch), showed greater infiltration of IL-31(+) cells in

the dermis compared to SD without severe itch, but no statistical significance was found (t-test, P

= 0.16) (Figure 1a).

Next, we investigated the cellular sources of IL-31. We detected a massive dermal

infiltrate comprising various types of immune cells (Figures 1a and 3). Most commonly among

them were CD68(+) macrophages, with the number of these cells significantly increased in SD

lesions compared to healthy controls (t-test, P = 0.0016) (Figure 1a). Therefore, we then

examined IL-31 expression by macrophages. As expected, the majority of IL-31(+) cells were

CD68(+) cells. In addition, the number of IL-31(+)/CD68(+) cells was significantly associated

with the presence of severe itch while the number of CD68(+) cells was approximately the same

in patients with and without severe itch (average ratio of IL-31(+)/CD68(+) cells to CD68(+)

cells was 88.2% in patients with severe itch vs 46.5% in patients without severe itch; t-test, P =

0.0096) (Figure 1b).

Macrophages are generally divided into two groups: M1 and M2 (Wang et al. 2014). Our

group previously reported that M2 macrophages are capable of generating IL-31 (Hashimoto et al.

2019b). Thus, we examined the expression of an M2 marker, CD163, by CD68(+) cells. The

6
number of CD163(+)/CD68(+) cells was increased in SD with severe itch compared with SD

without severe itch (t-test, P = 0.004) (Figure 1c).

β
Lesional expression of IL-31RA and OSMRβ

Both epidermal and dermal expression of IL-31RA were increased in SD lesions

compared to healthy controls (t-test, P < 0.0005 and P = 0.005, respectively), but were not

associated with the presence of severe itch (t-test, P = 0.74 and 0.58, respectively). OSMRβ

expression in the epidermis and the dermis was not enhanced in SD lesions compared to healthy

skin (t-test, P = 0.95 and 0.086, respectively) and was not associated with the presence of severe

itch (t-test, P = 0.18 and 0.78, respectively) (Figure 2).

Th2 and Th17 immune responses are predominant in SD lesions and correlated with IL-31

expression by macrophages

M2-macrophage skewing is closely related with Th2 immunity (Wang et al. 2014). The

number of dermal cells expressing C-C chemokine receptor type 4 (CCR4), which is

preferentially expressed by Th2 cells, increased in SD lesions compared to healthy controls (t-test,

P = 0.0026) and correlated with the number of CD68(+)/IL-31(+) cells (Spearman’s correlation, r

= 0.453, P = 0.020). The number of IL-17(+) cells was also increased in SD lesions compared to

healthy skin (t-test, P = 0.0045) and correlated with the number of CD68(+)/IL-31(+) cells

(Spearman’s correlation, r = 0.405, P = 0.040). In contrast, the number of cells that expressed

C-X-C chemokine receptor type 3 (CXCR3), a preferential marker for Th1 cells, did not change

between SD and healthy controls (t-test, P = 0.698) and was not correlated with the number of

CD68(+)/IL-31(+) cells (Spearman’s correlation, r = 0.326, P = 0.104), even though there was a

7
difference in the number of CXCR3(+) cells between SD lesions with severe itch and those

without severe itch (t-test, P = 0.036).

Basophils and eosinophils are involved in Th2 immunity (Hashimoto and Satoh 2018).

The number of dermal basophils was increased in SD lesions compared to heathy controls (t-test,

P <0.0005) and correlated with the number of CD68(+)/IL-31(+) cells (Spearman’s correlation, r

= 0.603, P = 0.001). The number of dermal eosinophils was neither increased in SD compared to

healthy controls (t-test, P = 0.106) nor correlated with the number of CD68(+)/IL-31(+) cells

(Spearman’s correlation, r = 0.145, P = 0.481) (Figure 3).

Periostin, substance P, and hemosiderin, but not TSLP, are correlated with IL-31 expression

by macrophages

Thymic stromal lymphopoietin (TSLP) and periostin promote M2 skewing (Furudate et

al. 2016; Han et al. 2013) and IL-31 generation from M2 macrophages (Hashimoto et al. 2019b).

Epidermal expression of TSLP was not enhanced in SD lesions compared to healthy controls

(t-test, P = 0.182). It was also not associated with the presence of severe itch (t-test, P = 0.355) or

with the number of CD68(+)/IL-31(+) cells (Spearman’s correlation, r = 0.213, P = 0.295). In

contrast, dermal deposition of perisotin was increased in SD lesions compared to healthy controls

(t-test, P <0.001) and correlated with the number of CD68(+)/IL-31(+) cells (Spearman’s

correlation, r = 0.552, P = 0.003), but was not directly related to the presence of severe itch (t-test,

P = 0.803).

SP and hemosiderin are also capable of promoting M2 skewing (Leal et al. 2015; Lim et

al. 2017; Rubio-Navarro et al. 2015). The number of dermal SP(+) cells and dermal deposition of

hemosiderin were increased in SD lesions compared to healthy controls (t-test, P <0.001 and P =

8
0.005, respectively) and correlated with the number of CD68(+)/IL-31(+) cells (Spearman’s

correlation, r = 0.463, P = 0.017 and r = 0.506, P = 0.008, respectively), but was not related to the

presence of severe itch (t-test, P = 0.802 and 0.859, respectively) (Figure 4).

CD68(+) macrophages express NK1R

SP exerts its function mainly, but not exclusively, through its receptor NK1R (Azimi et

al. 2017; Ständer and Yosipovitch 2019). SP stimulates NK1R expressing cells, resulting in the

release of additional itch mediators and evoking itch (Yosipovitch et al. 2018). Epidermal and

dermal expression of NK1R was enhanced in SD compared to healthy controls (t-test, P = 0.001

and P < 0.001, respectively) but was not associated with the presence of severe itch (t-test, P =

0.483 and 0.559, respectively) (Figure 5a). Of note, almost all CD68(+) macrophages expressed

NK1R (Figure 5b).

Skin innervation and other itch mediators

Intraepidermal nerve fibers (IENF) are involved in itch (Pereira et al. 2016). We found

reduced IENF density in SD lesions compared to healthy controls (t-test, P = 0.010) but no

association between IENF density and the presence of severe itch (t-test, P = 0.28).

Mast cells can release various itch mediators including histamine, prostaglandins, and

proteases (Steinhoff et al. 2018). The number of dermal mast cells did not change between SD

and healthy controls, or between SD with severe itch and SD without severe itch.

PAR-2 is a representative receptor for non-histaminergic itch (Steinhoff et al. 2003).

Epidermal expression of PAR-2 was not enhanced in SD compared to healthy controls (t-test, P =

9
0.734) and was reduced in SD without severe itch compared to SD with severe itch (t-test, P =

0.022).

TRPA1 and TRPV1 are ion channels that are involved in itch (Kittaka and Tominaga

2017). Epidermal expression of TRPA1 and TRPV1 was neither enhanced in SD compared to

healthy controls (t-test, P = 0.071 and 0.283, respectively) nor related to the presence of severe

itch (t-test, P = 0.375 and 0.139, respectively) (Supplemental Figure S1).

Murine peritoneal macrophages generate IL-31 in response to combination of substance P,

periostin, and red blood cell lysate

To elucidate the mechanisms of IL-31-generation from macrophages, we employed an ex

vivo stimulation test with murine peritoneal macrophages (pM) (Hashimoto et al. 2019b). pM

were stimulated with substance P, periostin, and/or RBC lysate, which represents hemosiderin.

Stimulation with SP alone did not significantly increase IL-31 mRNA expression in pM

(Supplemental Figure S2). In contrast, stimulation with periostin alone or periostin plus SP

significantly increased IL-31 mRNA expression. Of note, stimulation with the combination of SP,

periostin, plus RBC lysate dramatically enhanced IL-31 mRNA expression (Figure 6a). Protein

expression of IL-31 was confirmed with flow cytometric analysis. Flow cytometric analysis also

revealed that MOMA-2(+)/IL-31(+) pM express an M2 marker arginase-1, indicating that M2

macrophages generate IL-31 in response to a combination of SP, periostin and hemosiderin

(Figure 6b, c).

10
DISCUSSION

The present data showed that dermal IL-31 appeared to play a significant role in itch in

SD. Notably, the majority of IL-31 expressing cells were CD68(+) macrophages.

It is generally established that IL-31 is generated mainly by activated Th2 cells (Dillon

et al. 2004; Sonkoly et al. 2006). Other cellular types are also capable of generating IL-31,

including macrophages (Marquardt et al. 2011), eosinophils (Kunsleben et al. 2015), mast cells

(Niyonsaba et al. 2010), basophils (Raap et al. 2017), and keratinocytes (Nattkemper et al. 2016).

Among these, macrophages are reported to be the main cellular source in human skin lesions of

scabies (Hashimoto et al. 2019b), polymorphic light eruption (Patra et al. 2019), and AD (Kato et

al. 2014).

In SD lesions with severe itch, the majority of CD68(+)/IL-31(+) macrophages

expressed CD163, indicating they were M2 macrophages. In SD lesions without severe itch, a

smaller population of CD68(+) macrophages expressed CD163. These findings are congruent

with our previous study in scabies lesions with severe itch, which showed that dermal

accumulating M2 macrophages are the main cellular sources of IL-31 (Hashimoto et al. 2019b).

M2-macrophage polarization is promoted by Th2 immunity (Wang et al. 2014). We

found that a significant number of CCR4(+) Th2 cells and basophils infiltrated SD lesions.

Dermal deposition of periostin, a Th2-related protein (Masuoka et al. 2012), was also enhanced.

These findings indicate Th2 immunity-predominance in SD lesions. Even though these factors

were not directly associated with the presence of itch, they were significantly correlated with the

number of CD68(+)/IL-31(+) cells.

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 Interestingly, the number of IL-17(+) cells was increased in SD lesions, indicating Th17

immunity is also predominant in SD. The number of IL-17(+) cells was not directly related to the

presence of itch, but it was significantly correlated with the number of CD68(+)/IL-31(+) cells.

IL-17 is reported to promote M2 macrophage skewing (Nakai et al. 2017), and M2 macrophages

can promote Th17 cell-expansion (Haribhai et al. 2016; Mao et al. 2016). Thus, Th17 immunity

is likely linked to M2 macrophage skewing.

The number of SP(+) cells and amount of dermal deposition of hemosiderin were not

directly associated with itch, but were significantly correlated with the number of

CD68(+)/IL-31(+) macrophages. Mechanisms of overexpression of SP and periostin in SD are

unclear. SP is expressed by peripheral nerve fibers and various cell types including macrophages

(Marriott and Bost 2000) and T cells (Lai et al. 1998). These cells are abundant in SD lesions and

can be sources of SP.

To confirm the ability of macrophages to generate IL-31, we conducted ex vivo

stimulation studies with murine pM. We previously demonstrated that canonical Th2 cytokines

IL-4 and IL-13 do not stimulate pM to secrete a significant amount of IL-31. Instead, periostin

promotes M2 skewing and induces IL-31 generation from pM (Hashimoto et al. 2019b). The

current study shows IL-31 mRNA expression was dramatically increased in response to the

combination of periostin, SP, plus RBC lysate. In contrast, SP or RBC lysate alone did not

increase expression. Of note, IL-31 producing macrophages expressed arginase-1, indicating they

were M2 macrophages. Both human and murine macrophages express SP receptor NK1R,

hemosiderin receptor CD163, and periostin receptor integrin αV (Hashimoto et al. 2019b;

Siebenhaar et al. 2007). Collectively, SP, hemosiderin, and periostin in SD lesions can be

essentially involved in IL-31 generation from M2 macrophages. The SP/NK1R pathway can also

12
be involved in the generation of IL-31 by other cell populations such as eosinophils (Hashimoto

et al. 2019a).

We also investigated other itch mediators and IENF density in SD. Decreased IENF

density is reported to be involved in chronic itch in many skin diseases e.g., AD, psoriasis, and

prurigo nodularis (Schuhknecht et al. 2011; Tan et al. 2019). IENF density was reduced also in

SD lesions. No significant changes were observed in the number of mast cells and in the

expression of PAR-2, TRPA1, and TRPV1 between SD and healthy controls. Therefore, we

conclude that these factors do not significantly contribute to itch in SD.

IL-31 directly stimulates peripheral nerve fibers through IL-31RA and OSMRβ

(Cevikbas et al. 2014). In addition, IL-31 stimulates IL-31RA expressing cells, including

macrophages, basophils, and keratinocytes, to enhance inflammation (Nakashima et al. 2018).

Our study showed overexpression of IL-31RA, but not OSMRβ, both in the epidermis and the

dermis. Targeting IL-31 itself or IL-31RA may be beneficial in treating itch in SD. Macrophages

could also be potential therapeutic targets, in addition to SP and NK1R, periostin, and

hemosiderin. However, these targets require further consideration. In wound sites, M2

macrophages contribute to wound healing through promotion of fibrosis and angiogenesis (Kim

and Nair 2019; Snyder et al. 2016). SD is often accompanied by wound and chronic venous

ulcers (Sundaresan et al. 2017). Inhibition of M2-skewing might result in delayed wound healing.

This point should be examined in future studies.

The key limitation of this study was lack of patients’ numerical rating scale scores for

itch and we were not able to calculate correlation between protein expression and itch severity.

However, this study provides insights into the pathophysiological mechanisms of itch in SD.

IL-31 and IL-31RA can be useful therapeutic targets for itch in SD.

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MATERIALS AND METHODS

Samples

Skin lesional biopsy specimens and clinical data were obtained from 20 patients with SD

(age, 42-93 years old; 5 males and 15 females) and from 6 non-itchy healthy control subjects (age,

25-62; 4 males and 2 females) at Tokyo Medical and Dental University Hospital and University

of Miami Hospital. Their diagnoses were confirmed based on clinical and histological findings.

This study was approved by the ethical committees of Tokyo Medical and Dental University

(#M2018-033; informed written consent was not required because specimens were de-identified)

and University of Miami (no IRB number; informed written consent was not required as tissue

was collected from a de-identified repository and considered non-human research). Based on the

presence of itch according to their medical information, subjects were divided into two groups:

with severe itch (n=9; ages, 44-93; 3 males and 6 females) and without severe itch (n=11; ages,

42-85; 2 males and 9 females). Severe itch was defined as scale 4 in the Likert scale from zero

(no itch) to 4 (the worst), and without severe itch was defined as scales zero to 3. All patients had

no other pruritic skin disorders.

Mice

Seven-week-old female C57BL/6 mice were obtained from Sankyo Lab Service (Tokyo,

Japan). Mice were maintained under specific-pathogen-free conditions in our animal facility. All

animal experiments were approved by the Institutional Animal Care and Use Committee of

Tokyo Medical and Dental University (Protocols A2018-299A, A2018-199A, A2018-298A, and

A2018-237A) and performed at the Tokyo Medical and Dental University.

14
Antibodies

Abs used in this study are listed in Supplementary Material.

Immunofluorescence and iron staining

Formalin-fixed paraffin-embedded samples (5-µm) were deparaffinized and pretreated

with DAKO target retrieval solution (Dako, Glostrup, Denmark) at 60ºC overnight. Slides were

then treated with phosphate buffer solution with 5% normal donkey serum and 0.2% Triton

X-100 for 2 hours at room temperature. Next, sections were incubated with primary Abs at 4ºC

overnight followed by reaction with Alexa Fluor 488- or 594-conjugated secondary Abs

(Molecular Probes, Eugene, OR). Then, samples were mounted with Vectashield with DAPI

(Vector Laboratories, Burlingame, CA). For hemosiderin detection, Prussian blue staining was

used.

Quantification

Photomicrographs were captured with CTR6000 microscope (Leica Microsystems,

Wetzlar, Germany) at 20X magnification and three images from each subject were analyzed. For

periostin detection, whole scanning images were analyzed. The epidermal expression and dermal

periostin deposition were measured as fluorescence intensity in arbitrary units (AU) normalized

by area and background fluorescence using Image J software (NIH, Bethesda, MD). Dermal

deposition of hemosiderin was calculated by dividing Prussian blue stain-reactive area by dermal

area in each photomicrograph. The number of dermal infiltrating cells was manually quantified.

IENF density was calculated by dividing the number of neuron specific marker β-tubulin III(+)

15
nerves crossing the dermo-epidermal junction by the length of the epidermis (Sanders et al.

2019).

Preparation of murine peritoneal macrophages

Peritoneal cells were collected from C57BL/6N mice, seeded at the concentration of

5×105/well in plates in RPMI-1640 complete medium supplemented with 10% FBS and 100

IU/mL penicillin-streptomycin, and incubated for 2 h at 37°C and 5% CO2. Then non-adherent

cells were washed out and the remaining adherent cells (>80% of macrophages) were incubated

with or without substance P (1 µM; Peptide institute, Osaka, Japan), recombinant periostin (20

ng/mL; eBioscience, San Diego, CA), and/or RBC lysates (5% volume of total medium volume).

After 24 h, the cells were subjected to total RNA extraction or flow cytometric analysis. RBC

lysates were prepared as follows: murine whole blood was collected and centrifuged, and serum

were removed. Then blood was frosted and thawed twice.

Real-time PCR

Total cellular RNA was extracted from cells using ISOGEN II (Nippon Gene Co., Tokyo,

Japan), reverse-transcribed with SuperScript™ IV VILO™ Master Mix (Thermo Fisher

Scientific), and then quantitative RT-PCR was performed by real-time monitoring of the increase

in fluorescence of SYBR Green dye (Brilliant SYBR Green QPCR Master Mix; Agilent

Technologies Japan, Ltd., Tokyo, Japan) using the AriaMx Real-Time PCR System (Agilent

Technologies). The primers used for PCR were 5′-TCGGTCATCATAGCACATCTGGAG-3′ and

5′-GCACAGTCCCTTTGGAGTTAAGTC-3′ for mouse IL-31; and

5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′ for mouse

16
GAPDH. mRNA expression levels were calculated by the comparative ∆∆CT method relative to

GAPDH.

Flow cytometric analyses

Single cell-suspensions of cultured pM were obtained using cell scrapers after fixation.

They were pretreated with anti-CD16/32 Ab (BioLegend, San Diego, CA) and labeled with

MOMA-2 PE conjugated Ab (BioRad, Hercules, CA), anti-ariginase-1 Ab (Abcam plc,

Cambridge, UK: ab92274), and anti-IL-31 Ab (abcam: ab102750) followed by reaction with

Alexa Fluor 647 anti-goat IgG- and Alexa Fluor 488 anti-rabbit IgG- secondary Abs (Abcam plc,

ab150131 and ab150061, respectively) using Intracellular Fix & Perm set (eBioscience). Then

they were analyzed with FACSCalibur cell analyzer (BD Biosciences, San Jose, CA).

Statistical analysis

All data are reported as mean + SD. In order to compare differences between two groups,

two-tailed, unpaired t-tests were used. For detecting correlation, we calculated Spearman’s rank

correlation coefficient (r) using a statistical software “EZR” (Kanda 2013). Statistical

significance was set at P < 0.05.

17
Data Availability Statement: Datasets related to this article can be found at

http://dx.doi.org/10.17632/bmmkj2psfz.1, an open-source online data repository hosted at

Mendeley Data.

COI: Dr. Yosipovitch is Scientific Board Member of Menlo, Trevi, Sienna, Sanofi, Regeneron,

Galderma, Pfizer, Novartis, Bayer, Kiniksa, Eli Lilly, and Ortho. Research support by Pfizer, Sun

Pharma, Leo, Menlo, Kiniksa. The other authors have no conflicts of interest.

Acknowledgments: The authors thank Ms. Chiyako Miyagishi at Tokyo Medical and Dental

University for technical assistance. This work was supported by a Japan Society for the

Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Young Scientists (B) (#17K16328) and

by an unrestricted fellowship grant from Menlo Therapeutics.

CRediT Statement (author contribution):

Conceptualization: TH, GY. Data curation: TH. Formal analysis: TH. Funding acquisition: TH,

HY, GY. Investigation, TH, CK, RF, SN, SS. Methodology: TH, LN. Project administration: TH,

LN, HY, GY. Resources: TH, LN, HY, GY. Supervision: LN, HY, GY. Validation: TH, GY.

Visualization: TH, GY. Writing (the draft): TH, Writing (review and editing): TH, CK, RF, SN,

SS, LN, HY, GY. All the authors have read the manuscript and have approved this submission.

18
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FIGURE LEGENDS

Figure 1. CD68(+) macrophages express IL-31 and correlate with itch in stasis dermatitis.

Representative images of stasis dermatitis (SD) lesions and healthy skin with quantification of

staining. (a) Epidermal expression of IL-31 was not enhanced in SD lesions. The number of

IL-31(+) cells and CD68(+) macrophages was increased in SD lesions. (b) The majority of

IL-31(+) cells expressed CD68. The number of CD68(+)/IL-31(+) cells was increased in SD

lesions and correlated with itch. (c) In stasis dermatitis with severe itch, CD68(+) macrophages

expressed an M2 macrophage marker CD163. The number of CD68(+)/CD163(+) cells was

increased in SD lesions and correlated with itch. Scale bar = 100 µm. * = P < 0.05, unpaired t-test.

NS = not significant. AU = arbitrary unit. Dotted lines indicate the dermo-epidermal junction.

Vertical bars indicate SD.

β
Figure 2. Expression of IL-31 receptor complex components, IL-31RA and OSMRβ

Representative images of stasis dermatitis (SD) lesions and healthy skin with quantification of

staining. Both epidermal and dermal expression of IL-31RA was enhanced in SD lesions, but did

not correlate with itch. Expression of OSMRβ in the epidermis and dermis was not increased in

SD lesions and did not correlate with itch. Scale bar = 100 µm. * = P < 0.05, unpaired t-test. NS

= not significant. AU = arbitrary unit. Dotted lines indicate the dermo-epidermal junction.

Vertical bars indicate SD.

Figure 3. Infiltration of immune cells in stasis dermatitis.

Representative images of stasis dermatitis (SD) lesions and healthy skin with quantification of

staining. The number of C-X-C chemokine receptor type 3 (CXCR3)(+) cells was not increased
24
in SD lesions and did not correlate with the number of CD68(+)/IL-31(+) cells. In contrast, the

number of C-C chemokine receptor type 4 (CCR4) (+) cells and IL-17(+) cells was increased in

SD lesions and correlated with the number of CD68(+)/IL-31(+) cells. The number of dermal

basophils, but not dermal eosinophils, was increased in SD lesions and correlated with the

number of CD68(+)/IL-31(+) cells. Scale bar = 100 µm. * = P < 0.05, unpaired t-test. NS = not

significant. r = Spearman’s rank correlation coefficient. Vertical bars indicate SD.

Figure 4. Periostin, hemosiderin, and substance P, but not TSLP, are correlated with the

presence of IL-31(+) macrophages

Representative images of stasis dermatitis (SD) lesion and healthy skin with quantification of

staining. Epidermal expression of TSLP was not enhanced in SD lesions and did not correlate

with the number of CD68(+)/IL-31(+) cells. Dermal deposition of both periostin and hemosiderin,

as well as the number of substance P(+) cells was increased in SD lesions and correlated with

CD68(+)/IL-31(+) cells. All aforementioned factors were not significantly higher in SD with

severe itch compared to SD without severe itch. Scale bar = 100 µm for TSLP and substance P, =

1 mm for Periostin, and = 200 µm for hemosiderin. * = P < 0.05, unpaired t-test. NS = not

significant. AU = arbitrary unit. Dotted lines indicate the dermo-epidermal junction. Vertical bars

indicate SD.

Figure 5. CD68(+) macrophages express neurokinin-1 receptor

Representative images of stasis dermatitis (SD) lesions and healthy skin with quantification of

staining. (a) Both epidermal expression of neurokinin-1 receptor (NK-1R) and the number of

dermal NK-1R(+) cells were increased in SD lesions but did not correlate with severe itch. (b)

25
CD68(+) cells expressed NK-1R. Scale bar = 100 µm. * = P < 0.05, unpaired t-test. NS = not

significant. AU = arbitrary unit. Dotted lines indicate the dermo-epidermal junction. Vertical bars

indicate SD.

Figure 6. Murine peritoneal macrophages generate IL-31 in response to a combination of

substance P, periostin, and RBC lysate ex vivo

Murine peritoneal macrophages were stimulated with substance P, periostin, and/or RBC lysate

ex vivo for 24 hours. (a) IL-31 mRNA expression was significantly increased in response to

periostin, substance P plus periostin, and a combination of substance P + periostin + RBC lysate.

(b, c) Flow cytometric analysis of intracellular IL-31 and arginase-1 in MOMA-2 gated murine

peritoneal macrophages without stimulation (medium-treated, non-stimulated macrophages) or in

response to substance P plus periostin plus RBC lysate (stimulated macrophages). Representative

results of at least two independent experiments are shown. Values represent mean + SD. * = P <

0.05, unpaired t-test, compared to medium-treated, non-stimulated macrophages. MFI = mean

fluorescence intensity. Arg-1 = arginase-1.

26
SUPPLEMENTARY MATERIAL

SUPPLEMENTARY MATERIALS AND METHODS

Antibodies

Antibodies (Abs) obtained from Abcam plc (Cambridge, UK) included: Anti-mast

cell tryptase (AAI; ab2378), IL-31 (ab102750), IL-31RA (ab113498), C-X-C chemokine

receptor type 3 (ab64714), C-C chemokine receptor type 4 (ab1669), IL-17 (ab79056),

CD68 (KP1; ab955), CD163 (ab182422), basophil (2D7; ab155577), Substance P

(ab106291), thymic stromal lymphopoietin (ab188766), periostin (ab14041), transient

receptor potential vanilloid 1 (ab3487), transient receptor potential ankyrin 1 (ab62053),

and arginase-1 (ab92274) Abs. Anti-neurokinin 1 receptor (PA3-301), protease activated

receptor 2 (sc-5597), β-tubulin III (Tuj1; Mo15013), major basic protein (MBP)

(NBP1-42140-1ml), oncostatin M receptor β (LS-B11477) Abs were obtained from

ThermoFisher Scientifics (Waltham, MA), Santa Cruz Biotechnology (Dallas, TX),

Neuromics (Edina, MN), Novus biologicals (Centennial, CO), and LifeSpan BioSciences

(Seattle, WA), respectively. R-Phycoerythrin-conjugated anti-MOMA-2 Ab (MCA519PE)

was purchased from Bio Rad Laboratories (Hercules, CA). Alexa Fluor 647 anti-goat IgG-

(ab150131) and Alexa Fluor 488 anti-rabbit IgG- (ab150061) secondary Abs were obtained

from Abcam plc.

1
2
Supplemental Figure S1. Epidermal nerve fibers, mast cells, PAR-2, and TRPA1 in

lesional skin of stasis dermatitis.

Representative images of stasis dermatitis (SD) lesions and healthy skin with quantification

of staining. Intraepidermal nerve fiber (IENF) density was reduced in SD lesions but did

not correlate with itch. The number of dermal mast cells was not increased and did not

correlate with itch in SD lesions. Epidermal expression of PAR-2, TRPA1, and TRPV1 was

not enhanced in SD lesions. Scale bar = 100 µm. Dotted lines indicate the dermo-epidermal

junction. * = P < 0.05, unpaired t-test. Vertical bars indicate SD. NS = not significant. AU =

arbitrary unit. PAR-2 = protease-activated receptor-2. TRPA1 = transient receptor potential

ankyrin-1. TRPV1 = transient receptor potential vanilloid-1.

Supplemental Figure S2. IL-31 mRNA expression of murine peritoneal macrophages

in response to substance P

Representative results of two independent experiments are shown. Values represent mean +

SD of three samples. NS = not significant, unpaired t-test, compared to non-treated

macrophages.