# Household laundry detergents disrupt barrier integrity and induce inflammation in mouse and human skin

Arturo O Rinaldi, Manru Li, Elena Barletta, Paolo d'Avino, Duygu Yazici, Yagiz Pat, Siobhan Ward, Daniel Burla, Ge Tan, Nima Askary, et al.

► To cite this version:

Arturo O Rinaldi, Manru Li, Elena Barletta, Paolo d'Avino, Duygu Yazici, et al. Household laundry detergents disrupt barrier integrity and induce inflammation in mouse and human skin. Allergy, 2023, 79 (1), pp.128-141. 10.1111/all.15891. hal-05045863

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
ORIGINAL ARTICLE

Atopic Dermatitis, Urticaria and Skin Disease

# Household laundry detergents disrupt barrier integrity and induce inflammation in mouse and human skin

Arturo O. Rinaldi<fnref n="1" /> ID | Manru Li<fnref n="1" /> | Elena Barletta<fnref n="1" /> ID | Paolo D'Avino<fnref n="1" /> | Duygu Yazici<fnref n="1" /> |

Yagiz Pat<fnref n="1" /> | Siobhan Ward<fnref n="1" />,<fnref n="2" /> | Daniel Burla<fnref n="1" /> | Ge Tan<fnref n="1" /> ID | Nima Askary<fnref n="3" /> |

Rasmus Larsson<fnref n="3" /> | Jeremy Bost<fnref n="3" /> | Huseyn Babayev<fnref n="1" /> | Raja Dhir<fnref n="4" /> |

Nicolas Gaudenzio<fnref n="5" />,<fnref n="6" /> | Mubeccel Akdis<fnref n="1" /> ID | Kari Nadeau<fnref n="7" /> ID | Cezmi A. Akdis<fnref n="1" />,<fnref n="2" /> ID |

Yasutaka Mitamura<fnref n="1" /> ID

Correspondence

Yasutaka Mitamura and Cezmi A. Akdis, Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Herman-Burchard Strasse 9, CH-7265 Davos, Wolfgang, Switzerland.
Email: yasutaka.mitamura@siaf.uzh.ch and cezmi.akdis@siaf.uzh.ch

Funding information

Genoskin SAS; SciBase AB; the Swiss National Foundation

Abstract

**Background:** Epithelial barrier impairment is associated with many skin and mucosal inflammatory disorders. Laundry detergents have been demonstrated to affect epithelial barrier function in vitro using air-liquid interface cultures of human epithelial cells.

**Methods:** Back skin of C57BL/6 mice was treated with two household laundry detergents at several dilutions. Barrier function was assessed by electric impedance spectroscopy (EIS) and transepidermal water loss (TEWL) measurements after the 4 h of treatments with detergents. RNA sequencing (RNA-seq) and targeted multiplex proteomics analyses in skin biopsy samples were performed. The 6-h treatment effect of laundry detergent and sodium dodecyl sulfate (SDS) was investigated on ex vivo human skin.

**Results:** Detergent-treated skin showed a significant EIS reduction and TEWL increase compared to untreated skin, with a relatively higher sensitivity and dose-response in EIS. The RNA-seq showed the reduction of the expression of several genes essential for skin barrier integrity, such as tight junctions and adherens junction proteins. In contrast, keratinization, lipid metabolic processes, and epidermal cell differentiation were upregulated. Proteomics analysis showed that the detergents treatment generally downregulated cell adhesion-related proteins, such as epithelial cell adhesion molecule and contactin-1, and upregulated proinflammatory proteins, such as interleukin 6 and interleukin 1 beta. Both detergent and SDS led to a significant decrease in EIS values in the ex vivo human skin model.

**Abbreviations:** CCL2, C-C motif chemokine ligand 2; CCL3, C-C motif chemokine ligand 3; Cdh, cadherins; Cldn, claudin; CLSTN2, calsyntenin-2; CNTN1, contactin-1; CXCL1, C-X-C motif chemokine ligand 1; Dbn1, drebrin 1; Dsg4, desmoglein 4; EIS, electric impedance spectroscopy; EPCAM, epithelial cell adhesion molecule; Flg, filaggrin; FSTL3, follistatin-related protein 3; Gja3, gap junction protein alpha 3; IL1B, interleukin 1beta; IL6, interleukin 6; ITGB6, integrin beta-6; Ivl, involucrin; Jam2, junctional adhesion molecule 2; Lor, loricrin; Magi1, membrane-associated guanylate kinase inverted 1; Pard3, par-3 family cell polarity regulator; Pkp2, plakophilin 2; SDS, sodium dodecyl sulfate; Sprr, small proline rich protein; Tchp, trichoplein keratin filament binding; TEWL, transepidermal water loss; Tjap1, tight junction-associated protein 1.

Arturo O. Rinaldi and Manru Li equally contributed to this study.

This is an open access article under the terms of the [Creative Commons Attribution-NonCommercial License](https://creativecommons.org/licenses/by-nc), which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2023 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.

<footnote n="1">¹Swiss Institute of Allergy and Asthma Research (SIAF), University Zurich, Davos, Switzerland</footnote>

<footnote n="2">²Christine Kühne - Center for Allergy Research and Education (CK-CARE), Davos, Switzerland</footnote>

<footnote n="3">³SciBase AB, Sundbyberg, Sweden</footnote>

<footnote n="4">⁴SEED, Inc, Co, Los Angeles, California, USA</footnote>

<footnote n="5">⁵Toulouse Institute for Infectious and Inflammatory Diseases (Infinity), INSERM UMR1291, CNRS UMR5051, University Toulouse III, Toulouse, France</footnote>

<footnote n="6">⁶Genoskin SAS, Toulouse, France</footnote>

<footnote n="7">⁷Department of Environmental Studies, Harvard T.H. Chan School of Public Health, Cambridge, Massachusetts, USA</footnote>
**Conclusion:** The present study demonstrated that laundry detergents and its main component, SDS impaired the epidermal barrier in vivo and ex vivo human skin. Daily detergent exposure may cause skin barrier disruption and may contribute to the development of atopic diseases.

KEYWORDS

electrical impedance spectroscopy, epithelial barrier, ex vivo human skin, laundry detergents, skin inflammation, transepidermal water loss

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GRAPHICAL ABSTRACT

Household laundry detergents led to reduced electrical impedance, indicating impaired skin barrier. RNA-seq and targeted multiplex protein analyses in skin biopsy demonstrated that laundry detergents disrupted several major pathways involved in the skin barrier and induced inflammation. Laundry detergents and SDS caused barrier damage in ex vivo human skin.

Abbreviations: ACVRL1, activin A receptor like type; CCL, C-C motif chemokine ligand; CLSTN2, calsyntenin-2; CNTN1, contactin-1; EPCAM, epithelial cell adhesion molecule; FASTL3, follistatin-related protein 3; IL1B, interleukin 1 beta; IL6, interleukin 6; PBS, phosphate-buffered saline; RNA-seq, RNA sequencing; SDS, sodium dodecyl sulfate

1 | INTRODUCTION

The impairment of the epithelial barrier function is associated
with various skin and mucosal allergic, metabolic, and autoimmune
disorders. ¹⁻⁶ The prevalence of these diseases has been markedly
increasing in the Western world since the 1960s and continues to
increase in developing countries. ⁷⁻⁹ Increasing evidence points to
environmental factors playing a key role. ⁸,¹⁰⁻¹² A growing num-
ber of studies indicated an epidemiological association between

professional and domestic cleaning agents and the increased
prevalence of asthma, rhinitis, and allergic contact dermatitis. ¹³⁻²⁶
Certain cleaning products were also associated with the risk of ex-
acerbating asthma symptoms. ²⁷,²⁸ Detergents can also be present
as residues on surfaces and remain on clothing after rinsing during
laundry, thus getting in contact with the skin and the respiratory
mucosa. ²⁹ Interestingly, the post-rinse doses have been shown to
affect the epithelial barriers. ²⁹ The rinse residual liquid contained
the same amount of sodium dodecyl benzene sulfonate (SDBS)
in 1:2500 diluted commercial laundry detergent. Therefore, we urgently need to improve our understanding of how exposure to even low doses of cleaning products may contribute to allergic disorders.

In air-liquid interface cultures of human bronchial epithelial cells, laundry detergents reduced the barrier function affecting the expression of tight junctions (TJs).<sup>29</sup> These intercellular junctions consist of transmembrane and cytoplasmic proteins, also known as scaffold proteins. They represent one of the major contributions to barrier function, sealing paracellular spaces between neighboring epithelial cells at the very apical side in the mucosa and at the level of stratum granulosum in the skin.<sup>1,30,31</sup> In the epidermis, in addition to TJs, the stratum corneum contributes to forming a physically stronger barrier than mucosal membranes due to the expression of filaggrin (Flg), loricrin (Lor), and involucrin (Ivl).<sup>32,33</sup>

The present study aims to evaluate the in vivo effects of household laundry detergents on skin barrier integrity, even used at low concentration. We treated mouse skin with different dilutions of two household laundry detergents, and we assessed the epidermal barrier function using electrical impedance spectroscopy (EIS), previously described by our group as a valid method to detect skin barrier function in vivo.<sup>34</sup> Even when diluted 10,000 times, the epicutaneous application of household laundry detergents caused a reduction in skin barrier after only 4 h, together with significant changes in skin barrier-related and proinflammatory molecules in skin transcriptome and proteome, most of which correlated with the skin barrier EIS and transepidermal water loss (TEWL) measurements. Moreover, we showed that household laundry detergent and sodium dodecyl sulfate (SDS) can induce human skin barrier dysfunction.

## 2 | METHODS

### 2.1 | Mice

C57BL/6 mice were purchased from Charles River (Sulzfeld, Germany). They were housed in a specific-pathogen-free environment at the AO Research Institute, Davos (Switzerland) and used for experiments at 8-12 weeks of age. All the animal experiments were carried out in accordance with Swiss law and approved by the animal experiment commission of the canton Graubünden, Switzerland. All methods were carried out in accordance with Declaration of Helsinki principles.

### 2.2 | Epicutaneous administration of laundry detergents

Mice were depilated on their back skin using an epilation cream (Veet, Reckitt Benckiser). Three days after depilation, 150 µL of diluted (1:100, 1:1000 and 1:10,000 with phosphate-buffered saline [PBS]) two household laundry detergents, named here detergents A and B were applied for 4 h by means of a skin patch (cotton gauze and

Leukotape® classic) (Figure 1A). These concentrations were defined because the rinse residual liquid includes a similar concentration of SDBS with 1:2500 diluted commercial laundry detergent.<sup>29</sup> In addition, detergents A and B are recommended to be used at 1:285 and 1:400 dilutions for the hand wash, respectively. Several time points were studied in preliminary experiments, and 4 h was selected as the most optimal time point. PBS was used as control and to dilute detergents. After defined time points, mice were euthanized and sections of treated skin were collected.

### 2.3 | Epicutaneous treatments on Ex vivo human skin

Ex vivo human skin samples (NativeSkin®) were purchased (GenoSkin Inc., www.genoskin.com). Anonymized human skin samples were obtained from donors that underwent abdominoplasty procedures and had given their written informed consent. Donors did not have any record of allergies or dermatological disorders and did not use corticosteroids. Full ethical approval for the study protocol was obtained from the French ethical research committee (Comité de Protection des Personnes), and authorization was given from the French ministry of Research. Adult human skin explants were obtained from three healthy female donors (32, 37, and 57 years old) and bio-stabilized for up to 7 days. All samples were provided as 15 mm diameter round natural human skin biopsies in proprietary custom built plastic inserts.

Detergent B was diluted at the concentrations of 1:200, 1:1000, 1:5000, and 1:25,000 (v/v), and SDS was diluted at the concentrations of 5, 1, 0.2, and 0.04 mg/mL (w/v). Ex vivo human skin samples were treated with 60 µL of diluted household laundry detergent B and SDS. Detergent, SDS, and PBS were applied on the surface of ex vivo human skin for 6 h. The surface of NativeSkin samples was washed out with PBS three times after the stimulation.

### 2.4 | EIS measurements

EIS measurements were performed using Nevisense® (SciBase) like as previous study.<sup>34</sup> EIS is the measure of a material's opposition to the flow of alternating currents at various frequencies. Specifically, tissue EIS values can reflect the pathophysiological status of the tissue. The system measures the electrical impedance at 35 different frequencies distributed between 1 kHz and 2.5 MHz at four depths in 10 permutations. The applied voltage and current are limited to 150 mV and 75 µA, respectively, and its safety was proven in humans.<sup>35</sup>

For each mouse, EIS measurements were performed on the treated back skin before and 4, 8, 24, 48, and 72 h after the treatment with detergents or PBS application. For each time point, measurements were made in triplicates.

For ex vivo human skin, the surface of NativeSkin samples was moistened with PBS and wiped off with sterilized cotton gauze
<figure><img src="image_3.png" /><figcaption>FIGURE 1 Four-hour exposure to detergents decreases EIS and increases TEWL in the mouse skin. (A) WT C57BL/6 mice were depilated on their back. Three days after depilation, 150 µL of 1:10,000, 1:1000, and 1:100 diluted two different household laundry detergents (A and B) and its control PBS were applied by means of a skin patch for 4 h. EIS (B, D) and TEWL (C, E) measurements were performed before treatment (0) and 4, 8, 24, 48, and 72 h after the treatment with detergent A (B, C) and detergent B (D, E). EIS values are expressed in kOhm at a frequency of 1000Hz and TEWL in g/m²h. Data are shown as mean ± SEM. The colors of the stars showing statistical significance correspond to the colors of the dilutions shown in the legend. Mann-Whitney U-test was used to compare differences between each dilution of detergent analyzed to PBS control at each time point. \* p*<.05, \*\* p*<.01, \*\*\* p*<.001, \*\*\*\* p*<.0001. EIS, electric impedance spectroscopy; PBS, phosphate-buffered saline; TEWL, transepidermal water loss.</figcaption></figure>
before each measurement. The electrical impedance was measured at 35 different frequencies distributed between 1kHz and 2.5 MHz at two depths in three permutations after the optimization. EIS measurements were performed at 0, 6, 12, and 24h after the treatment. For each time point, measurements were made in triplicates.

## 2.5 | TEWL measurement

TEWL was measured by using a "Delfin VapoMeter®" (Delfin Technologies), whose core is a sensitive humidity sensor within a closed chamber. Its sensor monitors the increase of relative humidity inside the chamber during the skin measurement and automatically calculates the evaporation rate value (g/m²h) from the relative humidity increase. TEWL and EIS measurements were always performed in the same room and at the same body site of the mice.

## 2.6 | RNA isolation, RNA-seq, and data analysis

Mouse skin sections were collected and stored in RNAlater (Agilent). Skin sections were transferred into the appropriate amount of Qiazol lysis buffer (Qiagen), homogenized using gentleMACS Dissociator (Miltenyi Biotec) and gentleMACS M tubes (Miltenyi Biotec), and the total RNA was isolated using the RNeasy Plus Universal Mini kit (QIAGEN). The quantity and quality of the isolated RNA were determined respectively with the NanoDrop 2000 Spectrophotometer (Thermo Fischer Scientific) and 2200 TapeStation Automated Electrophoresis System (Agilent Technologies), and samples with RNA integrity numbers greater than 7.0 were chosen for sequencing. Library preparation for RNA sequencing (RNA-seq) was performed with the TruSeq Stranded mRNA Sample Prep Kit (Illumina).

Gene-level abundances were quantified as fragments per kilobase million values. Differentially expressed genes were assessed with a generalized linear model with the quasi-likelihood test from the Bioconductor package edgeR (version 3.30.3). Genes showing a statistically significant difference of *p*-value less than .01 were included in this study. False-discovery rate is calculated and shown in Table S1. Clustering analysis was performed with the "ward.D2" clustering algorithm implemented in the "hclust" function of the R statistics package. Heatmap plots were obtained with the function "heatmap" implemented in the gplots R package. Gene Ontology (GO) enrichment analysis on gene sets was conducted by using the online platform tool MetaCore (https://portal.genego.com).

## 2.7 | Quantitative reverse transcription PCR

Quantitative reverse transcription PCR (qRT PCR) was performed as previously described.³⁴

Primers for qRT-PCR were described in Table S2.

## 2.8 | Multiplex proximity extension assay

Mouse skin sections were collected and transferred into the appropriate amount of pre-cooled T-PER™ Tissue Protein Extraction Reagent (Thermo Fisher Scientific), containing phosphatase inhibitor cocktail "PhosSTOP" (Roche) and protease inhibitor cocktail "Complete, Mini, EDTA-free" (Roche). Skin sections were then homogenized using gentleMACS Dissociator and gentleMACS M tubes, and protein content was quantified using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Obtained skin lysates were analyzed by a proximity extension assay (Olink). This technology uses oligonucleotide-labeled antibody probe pairs that bind to their respective targets. Only upon binding of both antibody pairs to their target, DNA reporter molecules bound to the antibodies can form a double-strand DNA that is amplified and quantified by a real-time polymerase chain reaction assay. "Olink® Target 96 Mouse Exploratory" panel has been used.

## 2.9 | Statistical analyses

Mann-Whitney *U*-test was used to compare differences between each dilution of detergent compared to PBS control at the same point in the electric impedance data (kOhm). One-way ANOVA test was used to compare differences between dilutions of detergent and SDS at the same point in the normalized EIS data. Correlations between EIS scores, proteins in serum and severity scores were evaluated by Spearman's correlation test. All the statistical analyses were performed with GraphPad Prism 8.3 (GraphPad Software). A *p*-value <.05 was considered statistically significant.

Olink data were imported in normalized protein expression format, processed by "OlinkRPackage" from Olink Proteomics (https://github.com/Olink-Proteomics/OlinkRPackage). The statistical comparison of protein levels between groups was performed with Bioconductor limma package (https://bioconductor.org/packages/limma/). The fold change and *p*-value were estimated by fitting a linear model for each protein. The results were summarized in a heatmap and a volcano plot, with the magnitude of change in x-axis and the significance level in y-axis.

# 3 | RESULTS

## 3.1 | Skin barrier damage induced by household laundry detergents: A sensitive measurement by EIS

First, to evaluate the effect of detergents on the skin barrier, we measured EIS and TEWL before and 4, 8, 24, 48, and 72h after the application. As shown in Figure 1B,D, a detergent dose-dependent reduction in EIS was detected already 4h following the application of both detergents A and B, indicating a clear weakening of the skin barrier function. The electric impedance measurements showed a significant difference even in 1:10,000 dilution at 8h in response to detergent A and between 4 and 24h in response to detergent
B. After 24h, we observed an overall increase in electrical impedance and a decrease in TEWL, suggesting restoring barrier function. The effect of detergent B on damaging the skin barrier appeared to be stronger compared to detergent A when we observed both EIS and TEWL at different time points. In contrast, TEWL was not able to distinguish between the two lowest doses of both detergents. TEWL showed defective skin barrier only in the 1 to 100 dilution of the detergents, but did not show a dose-response (Figure 1C,E). The 4-h treatment with 1:100 diluted detergent is a relatively higher dose compared to the daily usage and post-rinse remnant doses. In conclusion, household laundry detergents were damaging the skin barrier even at 1:10,000 dilution in 4h, and EIS was more sensitive than TEWL measurements when assessing skin barrier integrity decrease.

## 3.2 | Skin transcriptome changes after detergent exposure

To analyze the detergent effects on skin transcriptome, we isolated RNA 24h after applying 1:1000 diluted detergent and performed RNA sequencing (Table S1). Interestingly, both detergents A and B extensively and significantly affected the transcriptome (Figure 2A,B). Specifically, detergent A induced the upregulation

of 454 genes and detergent B the upregulation of 751 genes, 377 of which were shared with detergent A. In analyses of the downregulated genes, detergents A and B affected the expression of 383 and 817 genes, respectively, sharing 287 of them (Figure 2A). As observed previously with EIS and TEWL measurements, detergent B appeared to have a more substantial effect on the up or down regulation of the skin mRNA transcripts. Furthermore, the differentially expressed genes shared between A and B (Figure 2B) were analyzed for GO term enrichment to identify the biological processes altered by the laundry detergents (Figure 2C). Biological processes significantly enriched by downregulated and upregulated genes are mainly associated with general impairment of the genes of the skin, such as skin development, epidermal cell differentiation, cornification, extracellular matrix organization, and several different lipid metabolic processes, such as biosynthesis, lipid membranes, sphingolipids, fatty acids, and ceramides (Listed in Table S3).

## 3.3 | Detergents disturb the expression of skin barrier gene families and increase keratinization

We analyzed the differential expression of the five skin barrier gene families: TJs, gap junctions, desmosomes, adherens junctions,

<figure><img src="image_4.png" /><figcaption>**FIGURE 2** Skin transcriptome changes after detergent exposure. RNA isolated from skin biopsies 24h after 4h treatment with 1:1000 dilution of both detergents A and B was sequenced using NGS. (A) Venn diagrams illustrate the number and the intersection of significantly differentially expressed genes in comparison of both detergents with the control group. (B) Heat map shows the comparison of the significantly differentially expressed genes included in the intersection between A and B. (C) The most significant GO terms of biological processes associated with the differentially expressed genes shown in B are reported. The number close to the bars indicates the number of significant differentially expressed genes involved in the corresponding process. GO, Gene Ontology.</figcaption></figure>
and keratinization, which include the whole set of genes involved in keratinocyte-keratinocyte interaction and forming of the physical barrier of the epidermis. Overall, our data show significant downregulation of the major skin barrier genes (Figure 3). Genes encoding for transmembrane proteins in TJs, such as claudins (Cldn) 2, 7, 10, 14 and junctional adhesion molecule 2 (Jam2), as well as for scaffold proteins, such as membrane-associated guanylate kinase inverted 1 (Magi1), par-3 family cell polarity regulator (Pard3) and tight junction-associated protein 1 (Tjap1), were significantly downregulated by 4h exposure of 1000 times diluted detergents. In addition, many other genes encoding for proteins essential in the formation of other cellular junctions, such as cadherins (Cdh) 3, 6, 11, 22, and 24, gap junction protein alpha 3 (Gja3), desmoglein 4 (Dsg4), plakophilin 2 (Pkp2), trichoplein keratin filament binding (Tchp), and drebrin 1 (Dbn1) were found to be downregulated. Interestingly, many genes involved in keratinization, such as Flg, Ivl, Lor, and several small proline rich proteins (Sprr), were upregulated after detergent application in the RNA seq data and validated by qPCR (Figure S1), suggesting a response against the toxic substance exposure and initiation of the healing process. In conclusion, the data demonstrate a huge down regulation of inter keratinocyte connections and upregulation of genes that support keratinization in the stratum corneum.

Furthermore, we observed a significant positive correlation between the EIS values recorded 24h after detergent application and the expression of skin barrier-related genes at the same time point. Mice showing the strongest decrease in EIS after detergent treatment were the ones with the strongest downregulation in the expression of genes related to intercellular junctions (Figure 4; Figure S2A). As expected, when we analyzed TEWL, we observed inverse correlations with skin barrier-related genes, but the changes and correlations were not as significant as observed with EIS determinations (Figure S2B).

## 3.4 | Detergents cause a decrease in cell adhesion-related proteins and induce skin inflammation

Following these results to confirm the changes at the protein expression level, we followed an exciting approach. Protein extract from skin samples taken 24 and 72h after detergent application was analyzed using the multiplex proximity extension assay. After analysis of the biological processes defined by the GO terms, we observed a significant downregulation of proteins associated with cell adhesion (Figure 5; Figure S3). Specifically, the epithelial cell adhesion molecule (EPCAM), integrin beta-6 (ITGB6), contactin-1 (CNTN1), calsyntenin-2 (CLSTN2), follistatin-related protein 3 (FSTL3), fibronectin leucine-rich transmembrane protein 2 (FLRT2), caspase 3 (CASP3), activin A receptor like type 1 (ACVRL1), and WNT1 inducible signaling pathway protein 1 (WISP1) were downregulated by both detergents. The downregulation occurred

already after 24h (Figure 5A,B) and was still detectable after 72h (Figure 5C,D). Some of them, such as CNTN1 and CLSTN2, downregulated even in 1:10,000 dilution after 72h. Interestingly, the strongest downregulation was observed at a 1:1000 dilution at both time points, whereas the least diluted sample (1:100) appeared to increase the expression of the proteins, EPCAM, ITGB6, FSTL3, CASP3, and WISP1 (Figure 5), which is probably because of a more robust induction of skin inflammatory responses, its negative feedback mechanism and starting of healing processes in high dose detergent exposure. In addition, at a 1:100 dilution the two detergents, particularly detergent B, caused an induction of the interleukins 1b and 6 (IL-1b and IL-6), C-C motif chemokine ligand 2 and 3 (CCL2 and CCL3), C-X-C motif chemokine ligand 1 (CXCL1), and TGFB1 that are related to immune responses and leukocyte chemotaxis (Figure S4). There was no significant increase in the expression of these proteins at 1:10,000 dilutions. CCL2 and CCL3 were already significantly increased at 1:1000 dilutions. The data are reposed in the Tables S4 and S5.

## 3.5 | EIS can detect the human skin barrier damage induced by household laundry detergents and sodium dodecyl sulfate

We further investigated the effect of laundry detergent on human skin using ex vivo human skin model. We applied the laundry detergent B and one of the main compounds of common detergents and cleaners, SDS on the NativeSkin samples.³⁶ NativeSkin samples are ex vivo human skin biopsies from living human donors, which contain the full architecture of the human skin, including epidermis, dermis, skin appendages, and tissue-resident immune cells. NativeSkin samples were exposed to household laundry detergent B at concentrations of 1:25,000, 1:5000, 1:1000, and 1:200 (v/v), SDS at concentrations of 0.04, 0.2, 1, and 5 mg/mL (w/v), and PBS as control for 6h. To evaluate their effect on the skin barrier, we measured EIS at before treatment (0) and 6, 12, and 24h after the application (Figure 6A; Figure S5A). As shown in Figure 6B, the NativeSkins were collected from the abdomen of the three donors and the average EIS results at 0h were 52.76–111.47 (kΩ) (Figure 6B). To minimize the variation in each sample and donor, we normalized the data by the result at 0h in each well. Consistent with our in vivo data, detergent B showed reduction in EIS after 6h treatment in a dose-dependent manner. These significant reductions continued until 24h in the applications of higher three doses (Figure 6C; Figure S5B). Even the lowest dose (1:25,000) showed a significant reduction after the treatment for 6h (Figure 6C). As shown in the Figure 6D and Figure S5C, SDS demonstrated a dose-dependent decrease in EIS following the application, indicating a clear weakening of the skin barrier. Our data suggest that EIS can be useful for detecting damage on ex vivo human skin barrier induced by environmental insults. Apparently, SDS plays an essential role in the skin barrier dysfunction caused by laundry detergents.
<figure><img src="image_5.png" /><figcaption>**FIGURE 3** Detergents affect the expression of skin barrier-related genes. (A) Volcano plot shows the differentially expressed skin barrier-related genes in comparison with the untreated control group. Genes with a *p*-value of less than .01 are marked in red. (B) Heatmaps show the significant differential expression of skin barrier gene families: adherens junctions, tight junctions, gap junctions, desmosomes, and keratinization.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>FIGURE 4 EIS values positively correlate with the mRNA expression of skin barrier-related genes. Scatter plots show the correlation of EIS values recorded 24 h after detergent application and the expression of skin barrier-related genes at the same time point. For each plot, the gene name is reported in the title and the Spearman r coefficient and p-value are shown. EIS is expressed in kOhm at a frequency of 1000 Hz and the gene expression in FPKM (fragments per kilobase per million reads mapped). EIS, electric impedance spectroscopy.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>FIGURE 5 Detergents induce a decrease in cell adhesion-related proteins. Total proteins were extracted from skin biopsies taken 24 and 72 h after different doses of detergent application (1:100, 1:1000, and 1:10,000) and were analyzed by proteomic Olink (Uppsala, Sweden) multiplex assay using the "Olink® Target 96 Mouse Exploratory panel." For detergent A (A, C) and detergent B (B, D), heatmaps show the significant differential expression of genes, associated with cell adhesion, 24 h (A, B) or 72 h (C, D) after detergent application.</figcaption></figure>
<figure><img src="image_8.png" /></figure>

<table><thead><tr><th>Age</th><th>Sex</th><th>Area</th><th>EIS result at 1000Hz (kΩ) at 0h (Median)</th></tr></thead><tbody><tr><td>57</td><td>Female</td><td>Abdomen</td><td>56.04-177.91 (111.47)</td></tr><tr><td>37</td><td>Female</td><td>Abdomen</td><td>45.11-89.53 (62.189)</td></tr><tr><td>32</td><td>Female</td><td>Abdomen</td><td>40.58-76.40 (52.76)</td></tr></tbody></table>

<figure><img src="image_9.png" /><figcaption>FIGURE 6 Six-hour exposure to detergents and sodium dodecyl sulfate decreases EIS in the ex vivo human skin. (A) Ex vivo human skin (NativeSkin®) samples were applied on the surface with 60μL of 1:25,000, 1:5000, 1:1000, and 1:200 (v/v) diluted household laundry detergent B, 0.04, 0.2, 1, and 5mg/mL diluted SDS, and control PBS for 6h. (B) Samples were collected from abdomen of three female donors. The range and median of the data in kOhm at a frequency of 1000Hz at 0h were displayed. (C, D) EIS measurements were performed before and after the laundry detergent B and SDS treatment at indicated concentrations and time points. The data were normalized with the result at 0h in each sample. One-way ANOVA test was used to compare differences between each dilution and PBS control at each time point. Data are shown as mean ± SEM. The colors of the stars showing statistical significance correspond to the colors of the dilutions shown in the legend. *: p < 0.05, **: p < .01, ***: p < .001, ****: p < .0001. EIS, electric impedance spectroscopy; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate.</figcaption></figure>

4 | DISCUSSION

The present study suggests that both laundry detergents and its main component, SDS can directly impair the integrity of the skin barrier in both mouse and human. Considering the fundamental role of a defective skin or mucosal barrier in the development of allergic disorders, it was proposed in the epithelial barrier hypothesis that detergents might contribute to the rise in allergy prevalence described in the past few decades in industrialized countries.⁷,²⁹,³⁷⁻³⁹

The epicutaneous application of different dilutions of two household laundry detergents led to a significant decrease in skin barrier function in vivo, as shown by the reduction in EIS and the concomitant increase in TEWL. Our data confirm that both TEWL and EIS represent reliable biomarkers/indicators of the skin barrier function, with a higher sensitivity and dose-response in EIS.³⁴,⁴⁰ TEWL measurements represent a popular and non-invasive method to assess the skin barrier function.⁴¹ As a new contribution to the area, EIS has been recently described as a useful tool for detecting epidermal barrier function rapidly and robustly without being affected by environmental humidity in vivo, reflecting the pathophysiological

status of the tissue.³⁴,⁴² In a recent study, we reported that EIS is a useful tool for discriminating epithelial barrier in the skin of AD patients compared to healthy controls and following up on healing after the treatment.⁴³ In addition, EIS scores showed a significant inverse correlation with protein biomarkers, particularly chemokines, which were significantly upregulated in the serum of patients.⁴³ These biomarkers were significantly associated with inflammatory and chemokine pathways that were observed in the skin of the detergent-treated mouse. In this study, we observed that both detergents exposure at a 1:10,000 dilution reduced electrical impedance. These results demonstrate that even single stimulation with 1:10,000 diluted detergents shows barrier-damaging effects in vivo in mice.

The topical application of the two detergents substantially reduced the expression of several genes essential for skin barrier integrity. TJs-related genes were significantly downregulated. These junctional complexes seal together adjacent epithelial cells and regulate the passage of ions and solutes through the intercellular space, thus contributing considerably to the barrier function.⁴⁴ Specifically, the detergent application significantly downregulated genes encoding for TJs transmembrane
proteins, such as Cldn 2, 7, 10, and 14 and Jam2, and those encoding for TJs scaffold proteins, such as Magi1, Pard3, and Tjap1. Besides, they also downregulated the expression of several adherens junctions related genes, such as Cdh3, 6, 11, 22, and 24, and Nectin3. Cadherins and nec-tins are transmembrane proteins crucial in forming adherens junctions, a distinct type of intercellular junctions important for epithelial adhesion.⁴⁴ Moreover, detergents downregulated the expression of gap junctions and desmosomes. The former create strong connections between ad-jacent epithelial cells on the apical side, together with TJs and adherens junctions; the latter localize beneath the apical junction complex along the lateral cell membrane.⁴⁵,⁴⁶ In our study, detergents downregulated the expression of Gja3, Dbn1, Tchp, Dsg4, and Pkp2.⁴⁷⁻⁵⁰ Taken together, these results indicate that the detergents impair the epithelial compact-ness and integrity by affecting the expression of skin barrier molecules. It is worth mentioning that the expression of all the skin barrier-related genes mentioned above positively correlated with EIS. This observation confirms the detrimental effect of detergents and proves the usefulness of EIS as a method to detect skin barrier integrity in vivo.

Notably, many genes encoding the various proteins involved in keratinization were upregulated by detergent exposure. The stratum corneum-related genes Flg, Ivl, Lor, and Sprr1 and Sprr2 were upregu-lated 24h after detergent application. Consistent with this, we found lipid metabolic processes, epidermal cell differentiation, and epidermis development among the biological processes significantly enriched by upregulated genes. It has previously been reported that Flg is down-regulated by several inflammatory cytokines and that lack of Flg cor-relates with increased levels of IL-1β.⁵¹ Although the levels of IL-1β and IL-6 in the tissue were upregulated in our model, the upregulation in the keratinization process might happen as the compensation reaction for the damage induced by the detergents by strengthening the stra-tum corneum keratinization, hence its physical barrier.

In the in vivo experiments, the present study demonstrates that EIS can differentiate the skin barrier-damaging doses of the detergents from 1 to 100 until 1 to 10,000 times dilutions in 4h exposure. When analyzing the skin proteome, we observed that the detergents gener-ally downregulated cell adhesion molecules 24h after their application. EPCAM, ITGB6, CNTN1, CLSTN2, FSTL3, FLRT2, CASP3, ACVRL1, and WISP1 are cell adhesion-related proteins and were all downregulated by the two detergents. Some of them, such as CNTN1, ACVRL1, and CLSTN2, remained downregulated 72h after detergent exposure. The downregulation of cell adhesion pathways could also contribute to the disruptive effect of detergents on skin barrier integrity. Remarkably, the least diluted sample (1:100) appeared to increase the expression of sev-eral barrier proteins with proinflammatory cytokines and chemokines. These results may suggest that detergents exposure at a 1:100 dilution cause skin barrier disruption with skin inflammation and lead a negative feedback and possibly a healing response 24h after detergent exposure.

In the present study, we showed that EIS has a sensitive diag-nostic ability to discriminate epithelial barrier changes between treated and untreated ex vivo human skin. This new approach al-lowed us to observe in vivo-like response related with the strati-fied layers and compartments of epithelial barrier in human skin.

With this approach, we clearly demonstrated the laundry deter-gent and its key compound SDS impair the barrier in a mouse model and an entire human skin biopsy. We are regularly exposed to various sources of detergents, including the detergent residue on our clothes. A recent study showed that the detergent residue after a complete rinse had similar composition of detergent with 2500 diluted commercial detergent and toxic effects.²⁹ It means our clothes, even after rinsing, still include a certain amount of detergents. Our study mimicking the daily detergent exposure via clothes demonstrated significant barrier-damaging effects even with a 1 to 25,000 dilution of SDS and commercial detergent. In addition, detergent with 1 to 200, 1000, and 5000 dilutions induced significant reductions and those effects continued until 24h. This result suggested that several 100 diluted detergents for hand wash are harmful doses to the skin.

Therefore, the prolonged exposure of detergent residues in the daily clothes could progressively impair the epidermal barrier, leading to ex-acerbations of eczema and a higher risk of allergen sensitization. Clinical evidence suggests that cutaneous exposure to food proteins through a disrupted skin barrier might lead to allergic sensitization.⁵² In addition, increased expression of proinflammatory cytokines and chemokines by detergents causes tissue inflammation and further damage to skin in-tegrity. Therefore, it can be speculated that the prolonged exposure to even low doses of detergents could notably increase the risk of allergen sensitization by impairing the skin barrier. Similarly, many types of wash-ing detergents and household cleaning products might also exacerbate already established allergy disorders. The concentration and the type of mainly cellular toxic surfactants and enzymes are decisive for their in vivo toxicity.³⁹ The ingredient information is presented in Figure S6. Our study shows that detergent B exhibited a more pronounced effect. De-tergent B contains a higher concentration of anionic surfactant (15%-30%) compared to detergent A (5%-15%), which likely can be a reason for the detrimental effects on mouse skin. A detergent contains 30 to 70 different molecules and the chemistry to get rid of stains from material by softening water, changing surface tension by surfactants, and using enzymes to degrade proteins. There is no international standardization for the dose of these toxic substances. In addition, other daily products, such as toothpastes and dishwasher detergents, contain SDS. They may damage oral, esophageal, and gut epithelial barriers.⁵³⁻⁵⁵ These daily cleaning products may induce barrier dysfunction especially in infant and allergic patients.⁶,⁵⁶,⁵⁷ Daily exposure to the detergents may be an important environmental factor that impairs the skin barrier, leads to allergen sensitization, and contributes to other allergic diseases.

Despite the novel findings highlighted in this study, it has some limitations. Our study shows a single-time stimulation. Future re-search efforts should focus on the repeated and chronic effects. In addition, commercial detergents are mixture of many components for several different functions. We showed the effect of SDS as one of the most commonly used anionic surfactants. The data demon-strated here are highly relevant to demonstrate molecular effects and epithelial barrier damage as it correlates with the suggested hand wash and post-rinse remnant doses. We will investigate the
effects of other components in our future research. In addition, the comparison with soaps and other non-toxic candidates as well as ef- fects on filaggrin mutated subjects remain for future studies.

In conclusion, the present study shows that household laundry detergents can disrupt the skin barrier, at daily used dilutions and in only for 4 h in the mouse model. As the main culprit highly diluted SDS impairs the human skin barrier. Overall, our data indicate that laun- dry detergents can impact the epidermis integrity and suggest further research to evaluate the long-term effect of detergents on the skin barrier, particularly in patients with skin disturbances. As a defective barrier has a critical role in the pathogenesis of allergic disorders, the daily exposure to detergents may represent an important issue of pub- lic health and therefore requires investigation by regulatory authori- ties. Lastly, these data confirmed EIS as a useful method to detect skin barrier integrity in vivo and in ex vivo models.

## AUTHOR CONTRIBUTIONS

Conceptualization: Arturo O. Rinaldi, Cezmi A. Akdis, and Yasutaka Mitamura. Methodology: Arturo O. Rinaldi, Manru Li, Siobhan Ward, Daniel Burla, and Paolo D'Avino. Investigation: Arturo O. Rinaldi and Manru Li. Visualization: Arturo O. Rinaldi, Manru Li, Ge Tan, and Elena Barletta. Funding acquisition: Cezmi A. Akdis. Project admin- istration: Arturo O. Rinaldi. Supervision: Mubeccel Akdis, Duygu Yazici, Yagiz Pat, Nicolas Gaudenzio, Raja Dhir, Jeremy Bost, Kari Nadeau, Raja Dhir, Nicolas Gaudenzio, Cezmi A. Akdis, and Yasutaka Mitamura. Writing—original draft: Arturo O. Rinaldi, Cezmi A. Akdis, and Yasutaka Mitamura. Writing—review & editing: Cezmi A. Akdis and Yasutaka Mitamura.

## ACKNOWLEDGMENTS

We acknowledge the contribution of Ass. Professor StigOllmar (founder of SciBase) for his basic research within this area. We also thank Beate Rückert and Anja Heider for their technical assistance during the study. We thank Anna Głobińska for her help in prepar- ing the graphical abstract. We thank the AO Research institute for the in vivo experiments in their specific-pathogen-free environment. Yasutaka Mitamura is a member of the research consortium SKIN-TEGRITY.CH. Open access funding provided by Universitat Zurich.

## FUNDING INFORMATION

This study was funded by the Swiss National Foundation. SciBase AB contributed EIS instruments and electrodes for the study and contributed toward study funding. Genoskin SAS provided Native-Skin samples for the study with special discounts.

## CONFLICT OF INTEREST STATEMENT

AR and CA report a patent application on "Methods and medical devices for analyzing epithelial barrier function." NA, RL, JB, CA, and YM report a patent pending for the technology titled "Methods and apparatus for measuring electrical impedance and assessing biological conditions of tissue samples." NA, RL, and JB declare receiving personal fees from SciBase AB. RD is a cofounder and CEO in Seed, a biotechnology company. GN declares receiving personal fees from

Genoskin SAS. CA has received research grants from the Swiss National Science Foundation, European Union (EU CURE, EU Syn- Air-G), Novartis Research Institutes, (Basel, Switzerland), Stanford University (Redwood City, Calif), and SciBase (Stockholm, Sweden); is the Co-Chair for EAACI Guidelines on Environmental Science in Allergic diseases and Asthma; is on the Advisory Boards of Sanofi/ Regeneron (Bern, Switzerland, New York, USA), Stanford Univer- sity Sean Parker Asthma Allergy Center (CA, USA), Novartis (Basel, Switzerland), Glaxo Smith Kline (Zurich, Switzerland), Bristol-Myers Squibb (New York, USA), Seed Health (Boston, USA) and SciBase (Stockholm, Sweden); and is the Editor-in-Chief of Allergy. KN re- ports grants from National Institute of Allergy and Infectious Dis- eases (NIAID), National Heart, Lung, and Blood Institute (NHLBI), National Institute of Environmental Health Sciences (NIEHS), and Food Allergy Research & Education (FARE); stock options from Ig- Genix, Seed Health, ClostraBio, and ImmunelD; is Director of the World Allergy Organization Center of Excellence for Stanford, Advi- sor at Cour Pharma, Consultant for Excellergy, Red tree ventures, Eli Lilly, and Phylaxis, Cofounder of Before Brands, Alladapt, Lati- tude, and IgGenix; and National Scientific Committee member at Immune Tolerance Network (ITN), and National Institutes of Health (NIH) clinical research centers, outside the submitted work; patents include, "Mixed allergen composition and methods for using the same," "Granulocyte-based methods for detecting and monitoring immune system disorders," and "Methods and Assays for Detecting and Quantifying Pure Subpopulations of White Blood Cells in Im- mune System Disorders." YM, ML, EB, PD, DY, YD, SW, DB, GT, HB, and MA have nothing to declare within the scope of this work.

## DATA AVAILABILITY STATEMENT

The accession number for the RNA sequencing data reported in this paper is Gene Expression Omnibus (GEO): GSE234841.

## ORCID

Arturo O. Rinaldi https://orcid.org/0000-0001-8967-3866

Elena Barletta https://orcid.org/0000-0002-8431-4140

Ge Tan https://orcid.org/0000-0003-0026-8739

Mubeccel Akdis https://orcid.org/0000-0003-0554-9943

Kari Nadeau https://orcid.org/0000-0002-2146-2955

Cezmi A. Akdis https://orcid.org/0000-0001-8020-019X

Yasutaka Mitamura https://orcid.org/0000-0001-6389-9285

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SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

How to cite this article: Rinaldi AO, Li M, Barletta E, et al. Household laundry detergents disrupt barrier integrity and induce inflammation in mouse and human skin. *Allergy*. 2024;79:128-141. doi:[10.1111/all.15891](https://doi.org/10.1111/all.15891)