# Dupilumab prevents nasal epithelial function alteration by IL-4 in vitro: Evidence for its efficacy

Maxime Fieux, Florent Carsuzaa, Yvan Bellanger, Sophie Bartier, Virginie Fournier, Jean Claude Lecron, Matthieu Bainaud, Bruno Louis, Stéphane Tringali, Xavier Dufour, et al.

► To cite this version:

Maxime Fieux, Florent Carsuzaa, Yvan Bellanger, Sophie Bartier, Virginie Fournier, et al. Dupilumab prevents nasal epithelial function alteration by IL-4 in vitro: Evidence for its efficacy. International Forum of Allergy and Rhinology, 2024, Online ahead of print. 10.1002/alr.23343. inserm-04546685

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# Dupilumab prevents nasal epithelial function alteration by IL-4 in vitro: Evidence for its efficacy

Maxime Fieux, Florent Carsuzaa, Yvan Bellanger, Sophie Bartier, Virginie Fournier, Jean Claude Lecron, Matthieu Bainaud, Bruno Louis, Stéphane Tringali, Xavier Dufour, et al.

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

## To cite this version:

Maxime Fieux, Florent Carsuzaa, Yvan Bellanger, Sophie Bartier, Virginie Fournier, et al. Dupilumab prevents nasal epithelial function alteration by IL-4 in vitro: Evidence for its efficacy. International Forum of Allergy and Rhinology, 2024, 14 (8), Online ahead of print. 10.1002/alr.23343. inserm-04546685

HAL Id: inserm-04546685

https://inserm.hal.science/inserm-04546685v1

Submitted on 15 Apr 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
ORIGINAL ARTICLE

International Forum of
Allergy
&Rhinology

# Dupilumab prevents nasal epithelial function alteration by IL-4 in vitro: Evidence for its efficacy

Maxime Fieux MD, PhD<sup>1,2,3,4 ID</sup>

Florent Carsuzaa MD<sup>5,6 ID</sup>

Yvan Bellanger MD<sup>3,4,7</sup>

Sophie Bartier MD<sup>3,4,7,8 ID</sup>

Virginie Fournier MLT<sup>3,4</sup>

Jean Claude Lecron PhD<sup>5,9</sup>

Matthieu Bainaud MD<sup>5,9</sup>

Bruno Louis PhD<sup>3,4 ID</sup>

Stéphane Tringali MD, PhD<sup>1,2,10</sup>

Xavier Dufour MD, PhD<sup>5,6</sup>

André Coste MD, PhD<sup>3,4,7</sup>

Laure Favot PhD<sup>5</sup>

Emilie Bequignon MD, PhD<sup>3,4,7</sup>

Correspondence

Maxime Fieux, Hospices Civils de Lyon,
Centre Hospitalier Lyon Sud, Service
d'ORL, d'Otoneurochirurgie et de
Chirurgie Cervico-Faciale, Pierre-Bénite,
Lyon F-69310, France.

Email: maxime.fieux@chu-lyon.fr

Florent Carsuzaa is a co-first author.

Laure Favot is a co-last author.

Funding information

la Société Française

d'Oto-Rhino-Laryngologie et de Chirurgie

Cervico-Faciale (SFORL); le Collège

Français d'Oto-Rhino-Laryngologie et de

Chirurgie Cervico-Faciale

(CFORL&CCF); Amplifon

Abstract

**Background:** Chronic rhinosinusitis with nasal polyp (CRSwNP) is a typical type 2 inflammation involving interleukin (IL)-4 and IL-13. Dupilumab is a fully human monoclonal antibody targeting IL-4 receptor α subunit, thereby blocking signaling by both cytokines. Our hypothesis was that IL-4 and IL-13, by inducing a severe epithelial dysregulation, are involved in CRSwNP pathogenesis. This study aimed to evaluate the in vitro direct effect of IL-4, IL-13, and dupilumab on nasal epithelial functions.

**Methods:** Nasal polyps and control mucosa from 28 patients, as well as human nasal epithelial cells (HNEC) from 35 patients with CRSwNP were used. Three major epithelial functions were investigated: the epithelial barrier function (characterized by transepithelial electrical resistance measurements and tight junction protein expression), the ciliary motion (characterized by the ciliary beating efficiency index), and wound healing (characterized by the wound repair

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

© 2024 The Authors. *International Forum of Allergy & Rhinology* published by Wiley Periodicals LLC on behalf of American Academy of Otolaryngic Allergy and American Rhinologic Society.

<footnote n="1"><sup>1</sup>Hospices Civils de Lyon, Centre Hospitalier Lyon Sud, Service d'ORL, d'Otoneurochirurgie et de Chirurgie Cervico-Faciale, Pierre Bénite, France</footnote>

<footnote n="2"><sup>2</sup>Faculté de Médecine et de Maïeutique Lyon Sud-Charles Mérieux, Université de Lyon, Université Lyon 1, Lyon, France</footnote>

<footnote n="3"><sup>3</sup>CNRS EMR 7000, Créteil, France</footnote>

<footnote n="4"><sup>4</sup>INSERM, IMRB, Univ Paris Est Créteil, Créteil, France</footnote>

<footnote n="5"><sup>5</sup>Laboratoire Inflammation Tissus Epithéliaux et Cytokines, UR15560, Université de Poitiers, Poitiers, France</footnote>

<footnote n="6"><sup>6</sup>Service ORL, Chirurgie Cervico-Maxillo-Faciale et Audiophonologie, Centre Hospitalier Universitaire de Poitiers, Poitiers, France</footnote>

<footnote n="7"><sup>7</sup>Centre Hospitalier Intercommunal de Créteil, Service d'Oto-Rhino-Laryngologie et de Chirurgie Cervico-Faciale, Créteil, France</footnote>

<footnote n="8"><sup>8</sup>Service d'ORL, de Chirurgie Cervico Faciale, Hôpital Henri-Mondor, Assistance Publique des Hôpitaux de Paris, Créteil, France</footnote>

<footnote n="9"><sup>9</sup>Service Immunologie et Inflammation, Centre Hospitalier Universitaire de Poitiers, Poitiers, France</footnote>

<footnote n="10"><sup>10</sup>UMR 5305, Laboratoire de Biologie Tissulaire et d'Ingénierie Thérapeutique, Institut de Biologie et Chimie des Protéines, CNRS, Université Claude Bernard Lyon 1, Lyon, France</footnote>
ORIGINAL ARTICLE

International Forum of
Allergy
&Rhinology

# Dupilumab prevents nasal epithelial function alteration by IL-4 in vitro: Evidence for its efficacy

Maxime Fieux MD, PhD<sup>1,2,3,4 ID</sup>

Florent Carsuzaa MD<sup>5,6 ID</sup>

Yvan Bellanger MD<sup>3,4,7</sup>

Sophie Bartier MD<sup>3,4,7,8 ID</sup>

Virginie Fournier MLT<sup>3,4</sup>

Jean Claude Lecron PhD<sup>5,9</sup>

Matthieu Bainaud MD<sup>5,9</sup>

Bruno Louis PhD<sup>3,4 ID</sup>

Stéphane Tringali MD, PhD<sup>1,2,10</sup>

Xavier Dufour MD, PhD<sup>5,6</sup>

André Coste MD, PhD<sup>3,4,7</sup>

Laure Favot PhD<sup>5</sup>

Emilie Bequignon MD, PhD<sup>3,4,7</sup>

**Correspondence**

Maxime Fieux, Hospices Civils de Lyon,
Centre Hospitalier Lyon Sud, Service
d'ORL, d'Otoneurochirurgie et de
Chirurgie Cervico-Faciale, Pierre-Bénite,
Lyon F-69310, France.

Email: maxime.fieux@chu-lyon.fr

Florent Carsuzaa is a co-first author.

Laure Favot is a co-last author.

**Funding information**

la Société Française

d'Oto-Rhino-Laryngologie et de Chirurgie
Cervico-Faciale (SFORL); le Collège
Français d'Oto-Rhino-Laryngologie et de
Chirurgie Cervico-Faciale
(CFORL&CCF); Amplifon

**Abstract**

**Background:** Chronic rhinosinusitis with nasal polyp (CRSwNP) is a typical type 2 inflammation involving interleukin (IL)-4 and IL-13. Dupilumab is a fully human monoclonal antibody targeting IL-4 receptor α subunit, thereby blocking signaling by both cytokines. Our hypothesis was that IL-4 and IL-13, by inducing a severe epithelial dysregulation, are involved in CRSwNP pathogenesis. This study aimed to evaluate the in vitro direct effect of IL-4, IL-13, and dupilumab on nasal epithelial functions.

**Methods:** Nasal polyps and control mucosa from 28 patients, as well as human nasal epithelial cells (HNEC) from 35 patients with CRSwNP were used. Three major epithelial functions were investigated: the epithelial barrier function (characterized by transepithelial electrical resistance measurements and tight junction protein expression), the ciliary motion (characterized by the ciliary beating efficiency index), and wound healing (characterized by the wound repair

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

© 2024 The Authors. *International Forum of Allergy & Rhinology* published by Wiley Periodicals LLC on behalf of American Academy of Otolaryngic Allergy and American Rhinologic Society.

<footnote n="1"><sup>1</sup>Hospices Civils de Lyon, Centre Hospitalier Lyon Sud, Service d'ORL, d'Otoneurochirurgie et de Chirurgie Cervico-Faciale, Pierre Bénite, France</footnote>

<footnote n="2"><sup>2</sup>Faculté de Médecine et de Maïeutique Lyon Sud-Charles Mérieux, Université de Lyon, Université Lyon 1, Lyon, France</footnote>

<footnote n="3"><sup>3</sup>CNRS EMR 7000, Créteil, France</footnote>

<footnote n="4"><sup>4</sup>INSERM, IMRB, Univ Paris Est Créteil, Créteil, France</footnote>

<footnote n="5"><sup>5</sup>Laboratoire Inflammation Tissus Epithéliaux et Cytokines, UR15560, Université de Poitiers, Poitiers, France</footnote>

<footnote n="6"><sup>6</sup>Service ORL, Chirurgie Cervico-Maxillo-Faciale et Audiophonologie, Centre Hospitalier Universitaire de Poitiers, Poitiers, France</footnote>

<footnote n="7"><sup>7</sup>Centre Hospitalier Intercommunal de Créteil, Service d'Oto-Rhino-Laryngologie et de Chirurgie Cervico-Faciale, Créteil, France</footnote>

<footnote n="8"><sup>8</sup>Service d'ORL, de Chirurgie Cervico Faciale, Hôpital Henri-Mondor, Assistance Publique des Hôpitaux de Paris, Créteil, France</footnote>

<footnote n="9"><sup>9</sup>Service Immunologie et Inflammation, Centre Hospitalier Universitaire de Poitiers, Poitiers, France</footnote>

<footnote n="10"><sup>10</sup>UMR 5305, Laboratoire de Biologie Tissulaire et d'Ingénierie Thérapeutique, Institut de Biologie et Chimie des Protéines, CNRS, Université Claude Bernard Lyon 1, Lyon, France</footnote>
rate) under various stimulations (IL-4, IL-13, and dupilumab). The main outcome was a significant change in epithelial functions following exposure to IL-4, IL-13, and dupilumab for 48 h in the basal media.

**Results:** IL-4 (1, 10, and 100 ng/mL) but not IL-13 induced a significant decrease in occludin and zonula-occludens protein expression, ciliary beating efficiency, and wound repair rate in HNEC. Dupilumab (0.04 mg/mL) had no effect on HNEC and specifically restored all epithelial functions altered when cells were exposed to a 48-h IL-4 stimulation.

**Conclusion:** Dupilumab, in vitro, restored epithelial integrity by counteracting the effect of IL-4 on the epithelial barrier (increased epithelial permeability, decreased ciliary beating efficiency, and decreased wound repair rate).

### KEYWORDS

chronic rhinosinusitis, ciliary motility, epithelial cell, paranasal sinus diseases

## 1 | INTRODUCTION

In western Europe, up to 11% of the population is affected by chronic rhinosinusitis with nasal polyps (CRSwNP),¹ an inflammatory disease of the airways² with a type 2 (T2) inflammatory pattern in 85% of CRSwNP cases.³ CRSwNP is characterized by epithelial damage including goblet cell metaplasia and decreased ciliated cell differentiation.² The nasal epithelium is an important mechanical barrier and responds to environmental insults by inducing the production of T2 promoting cytokines.⁴ To ensure their barrier function, neighboring human nasal epithelial cells (HNEC) are connected to one another via a tight junction (TJ) protein complex. In CRSwNP patients, loss of TJ function and/or expression has been observed⁵; the disruption of the epithelial barrier leads to a chronic inflammatory response that fails to resolve.⁶⁻⁹ T2 inflammation, epithelial dysfunction, and numerous cytokines appear to be key factors in the pathogenesis of CRSwNP and the formation of nasal polyps.²,¹⁰,¹¹

IL-4 and IL-13 are key T2 cytokines sharing a common receptor subunit (IL-4 receptor α subunit, IL-4Rα¹²). High levels of IL-4 and IL-13 have already been reported in the mucus of patients with CRSwNP,¹³,¹⁴ and both IL-4 and IL-13 are deeply involved in airway remodeling.¹⁵⁻²¹ IL-4 has been shown to reduce the expression of the TJ proteins zonula-occludens (ZO-1) and occludin,²² but these results remain debated as other authors did not find any impact of IL-4 on TJ.²³ IL-4 but not IL-13 was found to decrease wound repair rate (WRR) in vitro²⁴ and increase epithelial permeability.²³ Conversely, IL-4 and IL-13 were both shown to induce epithelial migration.²² Dupilumab, a fully human monoclonal antibody (mAb) that binds IL-4Rα, thereby blocking signaling by both cytokines,

has been shown to be effective in multiple T2 diseases (asthma, atopic dermatitis, and CRSwNP).²⁵,²⁶ IL-4 blockade potently prevents B-cell activation, immunoglobulin E (IgE) production, and pathogenic T-cell lung infiltration, while inhibition of IL-13 is required to prevent goblet cell metaplasia.²⁶ All these results support our main hypothesis that IL-4 and IL-13 are responsible for severe epithelial dysregulation, which can be measured by testing nasal epithelial functions. Moreover, by targeting IL-4Rα, dupilumab should have a positive impact on these same functions, explaining why it became the optimum treatment for T2 diseases. Nevertheless, the direct effect of dupilumab on the nasal epithelial barrier in vitro has never been evaluated.

The main aim of the present study was to evaluate the direct effect of IL-4, IL-13, and dupilumab on nasal epithelial functions in CRSwNP.

## 2 | MATERIALS AND METHODS

### 2.1 | Tissue and cell collection

First, the mRNA expression of IL-4, IL-13, as well as the protein expression of occludin were analyzed in nasal polyps and control mucosa (n = 28). For control mucosa, small-sized septal mucosa fragments were obtained from patients operated on for non-inflammatory and non-tumoral diseases (n = 14). Nasal polyps were taken from patients suffering from CRSwNP with or without comorbidities such as aspirin-exacerbated respiratory diseases (AERD; n = 14); no allergic fungal rhinosinusitis (AFRS) or CRS sin NP (CRSsNP) patients were included in this study. Tissues were frozen in liquid nitrogen
rate) under various stimulations (IL-4, IL-13, and dupilumab). The main outcome was a significant change in epithelial functions following exposure to IL-4, IL-13, and dupilumab for 48 h in the basal media.

**Results:** IL-4 (1, 10, and 100 ng/mL) but not IL-13 induced a significant decrease in occludin and zonula-occludens protein expression, ciliary beating efficiency, and wound repair rate in HNEC. Dupilumab (0.04 mg/mL) had no effect on HNEC and specifically restored all epithelial functions altered when cells were exposed to a 48-h IL-4 stimulation.

**Conclusion:** Dupilumab, in vitro, restored epithelial integrity by counteracting the effect of IL-4 on the epithelial barrier (increased epithelial permeability, decreased ciliary beating efficiency, and decreased wound repair rate).

### KEYWORDS

chronic rhinosinusitis, ciliary motility, epithelial cell, paranasal sinus diseases

## 1 | INTRODUCTION

In western Europe, up to 11% of the population is affected by chronic rhinosinusitis with nasal polyps (CRSwNP),¹ an inflammatory disease of the airways² with a type 2 (T2) inflammatory pattern in 85% of CRSwNP cases.³ CRSwNP is characterized by epithelial damage including goblet cell metaplasia and decreased ciliated cell differentiation.² The nasal epithelium is an important mechanical barrier and responds to environmental insults by inducing the production of T2 promoting cytokines.⁴ To ensure their barrier function, neighboring human nasal epithelial cells (HNEC) are connected to one another via a tight junction (TJ) protein complex. In CRSwNP patients, loss of TJ function and/or expression has been observed⁵; the disruption of the epithelial barrier leads to a chronic inflammatory response that fails to resolve.⁶⁻⁹ T2 inflammation, epithelial dysfunction, and numerous cytokines appear to be key factors in the pathogenesis of CRSwNP and the formation of nasal polyps.²,¹⁰,¹¹

IL-4 and IL-13 are key T2 cytokines sharing a common receptor subunit (IL-4 receptor α subunit, IL-4Rα¹²). High levels of IL-4 and IL-13 have already been reported in the mucus of patients with CRSwNP,¹³,¹⁴ and both IL-4 and IL-13 are deeply involved in airway remodeling.¹⁵⁻²¹ IL-4 has been shown to reduce the expression of the TJ proteins zonula-occludens (ZO-1) and occludin,²² but these results remain debated as other authors did not find any impact of IL-4 on TJ.²³ IL-4 but not IL-13 was found to decrease wound repair rate (WRR) in vitro²⁴ and increase epithelial permeability.²³ Conversely, IL-4 and IL-13 were both shown to induce epithelial migration.²² Dupilumab, a fully human monoclonal antibody (mAb) that binds IL-4Rα, thereby blocking signaling by both cytokines,

has been shown to be effective in multiple T2 diseases (asthma, atopic dermatitis, and CRSwNP).²⁵,²⁶ IL-4 blockade potently prevents B-cell activation, immunoglobulin E (IgE) production, and pathogenic T-cell lung infiltration, while inhibition of IL-13 is required to prevent goblet cell metaplasia.²⁶ All these results support our main hypothesis that IL-4 and IL-13 are responsible for severe epithelial dysregulation, which can be measured by testing nasal epithelial functions. Moreover, by targeting IL-4Rα, dupilumab should have a positive impact on these same functions, explaining why it became the optimum treatment for T2 diseases. Nevertheless, the direct effect of dupilumab on the nasal epithelial barrier in vitro has never been evaluated.

The main aim of the present study was to evaluate the direct effect of IL-4, IL-13, and dupilumab on nasal epithelial functions in CRSwNP.

## 2 | MATERIALS AND METHODS

### 2.1 | Tissue and cell collection

First, the mRNA expression of IL-4, IL-13, as well as the protein expression of occludin were analyzed in nasal polyps and control mucosa (n = 28). For control mucosa, small-sized septal mucosa fragments were obtained from patients operated on for non-inflammatory and non-tumoral diseases (n = 14). Nasal polyps were taken from patients suffering from CRSwNP with or without comorbidities such as aspirin-exacerbated respiratory diseases (AERD; n = 14); no allergic fungal rhinosinusitis (AFRS) or CRS sin NP (CRSsNP) patients were included in this study. Tissues were frozen in liquid nitrogen
before protein and nucleic acid extraction. The study was approved by an institutional review board (agreement number DC-20193746, December 2019).

## 2.2 | Primary culture of HNEC

Using primary cultures of HNEC at the air-liquid interface (ALI), the mRNA expression pattern of IL-4 receptors and the effect of IL-4, IL-13, and dupilumab on transepithelial electrical resistance (TEER), TJ protein expression, ciliary beating efficiency, and wound repair mechanisms were evaluated. To analyze TJ protein expression, two proteins of this complex, occludin and ZO-1, were chosen in this study.²⁷,²⁸

The nasal polyps from 35 patients operated on for CRSwNP were obtained and immediately placed in culture medium and transported to the laboratory for HNEC isolation and culture, as previously described by Coste et al.⁶ (Supporting Information). The approach used to establish primary cultures of HNEC at ALI was the same as that developed more than 20 years ago by our team.⁷,²⁹⁻³¹ As described previously, nasal polyps were taken from patients suffering from CRSwNP with or without comorbidities such as AERD (no AFRS or CRSsNP patients). All patients abstained from oral corticosteroids during the month before the surgery and had given informed consent (agreement by the local ethics committee, CPP IDF X 2016-01-01). The epithelial nature of ALI-cultured HNEC was confirmed by flow cytometry and cytokeratin 14 immunofluorescence analysis. After intense proliferation during the first week, the HNEC reach their differentiated stage during the third week of culture and were used herein from day 21 onwards.⁶,³¹

In order to test the effect of IL-4, IL-13, and dupilumab on epithelial function, the same various conditions were tested for each function (barrier, ciliary beating, repair): IL-4 alone, IL-13 alone, dupilumab alone, IL-4 with dupilumab, anti-IL-6 alone, and IL-4 with anti-IL-6. All were placed in the basal media for 48 h (referred as 48 h of cytokine exposure in Section 3); basal medium alone was used as control. IL-4 and IL-13 were tested at 1, 10, and 100 ng/mL concentrations, according to previous studies on epithelial cells.¹¹,²³,²⁴,³² Dupilumab was used at a concentration of 0.04 mg/mL, which is the equivalent concentration of that available in the tissue after a subcutaneous injection (Supporting Information). IL-6 is a multifunctional cytokine that plays an important role in inflammation and CRSwNP pathogenesis¹¹; the latter however does not target IL-4Rα. In order to assess whether the effect of dupilumab (an IgG4 of 147 kDa) was specific, a neutralizing monoclonal isotype antibody targeting human IL-6 was used (anti-IL-6, 145 kDa, Invivogen). This

mAb, called anti-IL-6, was used at the same concentration than dupilumab (0.04 mg/mL).

## 2.3 | IL-4 and IL-13 mRNA expression and occludin protein expression in nasal polyps and control mucosa

To evaluate IL-4 and IL-13 mRNA expression, total RNA from the nasal polyps and control mucosa was isolated using a NucleoSpin RNA II kit (Macherey-Nagel). Occludin protein expression was evaluated using Western blot. Details appear in Supporting Information.

## 2.4 | IL-4 receptor α expression in HNEC (mRNA)

To evaluate IL-4Rα mRNA expression in HNEC, total RNA from HNEC, after 21 days of culture, was isolated using a NucleoSpin RNA II kit (Macherey-Nagel; Supporting Information).

## 2.5 | Occludin protein expression in HNEC (Western blot)

To evaluate the effects of IL-4, IL-13, and dupilumab on epithelial barrier function, the in vitro expression of occludin was quantified using Western blot in HNEC (Supporting Information).

## 2.6 | ZO-1 protein expression in HNEC (immunolabeling)

To evaluate further the effects of IL-4, IL-13, and dupilumab on epithelial barrier function, after TEER measurements were performed (see below), labeling of actin and immunolabeling of ZO-1 were performed (Supporting Information). To focus on the TJ belt, we chose to acquire all images at the apical pole of the cell. Immunolabeling and labeling were performed three times, but only representative images are shown. In all conditions, the same parameters (labeling intensity and magnifications) for ZO-1 and actin expression were used to ensure that a qualitative analysis could be performed.

## 2.7 | TEER in HNEC (electrical resistance)

TEER provides a physical measure of the electrical resistance across the epithelial cell barrier and is widely used as
before protein and nucleic acid extraction. The study was approved by an institutional review board (agreement number DC-20193746, December 2019).

## 2.2 | Primary culture of HNEC

Using primary cultures of HNEC at the air-liquid interface (ALI), the mRNA expression pattern of IL-4 receptors and the effect of IL-4, IL-13, and dupilumab on transepithelial electrical resistance (TEER), TJ protein expression, ciliary beating efficiency, and wound repair mechanisms were evaluated. To analyze TJ protein expression, two proteins of this complex, occludin and ZO-1, were chosen in this study.²⁷,²⁸

The nasal polyps from 35 patients operated on for CRSwNP were obtained and immediately placed in culture medium and transported to the laboratory for HNEC isolation and culture, as previously described by Coste et al.⁶ (Supporting Information). The approach used to establish primary cultures of HNEC at ALI was the same as that developed more than 20 years ago by our team.⁷,²⁹⁻³¹ As described previously, nasal polyps were taken from patients suffering from CRSwNP with or without comorbidities such as AERD (no AFRS or CRSsNP patients). All patients abstained from oral corticosteroids during the month before the surgery and had given informed consent (agreement by the local ethics committee, CPP IDF X 2016-01-01). The epithelial nature of ALI-cultured HNEC was confirmed by flow cytometry and cytokeratin 14 immunofluorescence analysis. After intense proliferation during the first week, the HNEC reach their differentiated stage during the third week of culture and were used herein from day 21 onwards.⁶,³¹

In order to test the effect of IL-4, IL-13, and dupilumab on epithelial function, the same various conditions were tested for each function (barrier, ciliary beating, repair): IL-4 alone, IL-13 alone, dupilumab alone, IL-4 with dupilumab, anti-IL-6 alone, and IL-4 with anti-IL-6. All were placed in the basal media for 48 h (referred as 48 h of cytokine exposure in Section 3); basal medium alone was used as control. IL-4 and IL-13 were tested at 1, 10, and 100 ng/mL concentrations, according to previous studies on epithelial cells.¹¹,²³,²⁴,³² Dupilumab was used at a concentration of 0.04 mg/mL, which is the equivalent concentration of that available in the tissue after a subcutaneous injection (Supporting Information). IL-6 is a multifunctional cytokine that plays an important role in inflammation and CRSwNP pathogenesis¹¹; the latter however does not target IL-4Rα. In order to assess whether the effect of dupilumab (an IgG4 of 147 kDa) was specific, a neutralizing monoclonal isotype antibody targeting human IL-6 was used (anti-IL-6, 145 kDa, Invivogen). This

mAb, called anti-IL-6, was used at the same concentration than dupilumab (0.04 mg/mL).

## 2.3 | IL-4 and IL-13 mRNA expression and occludin protein expression in nasal polyps and control mucosa

To evaluate IL-4 and IL-13 mRNA expression, total RNA from the nasal polyps and control mucosa was isolated using a NucleoSpin RNA II kit (Macherey-Nagel). Occludin protein expression was evaluated using Western blot. Details appear in Supporting Information.

## 2.4 | IL-4 receptor α expression in HNEC (mRNA)

To evaluate IL-4Rα mRNA expression in HNEC, total RNA from HNEC, after 21 days of culture, was isolated using a NucleoSpin RNA II kit (Macherey-Nagel; Supporting Information).

## 2.5 | Occludin protein expression in HNEC (Western blot)

To evaluate the effects of IL-4, IL-13, and dupilumab on epithelial barrier function, the in vitro expression of occludin was quantified using Western blot in HNEC (Supporting Information).

## 2.6 | ZO-1 protein expression in HNEC (immunolabeling)

To evaluate further the effects of IL-4, IL-13, and dupilumab on epithelial barrier function, after TEER measurements were performed (see below), labeling of actin and immunolabeling of ZO-1 were performed (Supporting Information). To focus on the TJ belt, we chose to acquire all images at the apical pole of the cell. Immunolabeling and labeling were performed three times, but only representative images are shown. In all conditions, the same parameters (labeling intensity and magnifications) for ZO-1 and actin expression were used to ensure that a qualitative analysis could be performed.

## 2.7 | TEER in HNEC (electrical resistance)

TEER provides a physical measure of the electrical resistance across the epithelial cell barrier and is widely used as
an indicator of in vitro permeability.³³ TEER was measured using an epithelial volt ohm meter with an ENDOHM-12 electrode (World Precision Instruments) as described previously.³⁴ Only HNEC with a TEER > 200 Ω/cm² were used (Supporting Information).

## 2.8 | Ciliary beating efficiency in HNEC (high-speed video microscopy)

The effect of IL-4 and IL-13 on the ciliary beat frequency (CBF) of respiratory epithelium has already been described and has been shown to contribute to the impaired mucociliary clearance in vitro.³⁵ Although, CBF is not the best tool to evaluate ciliary beating efficiency, another one described by our team, the efficiency index of ciliary clearance was chosen for the present study.³⁶,³⁷ In order to evaluate further the effects of IL-4, IL-13, and dupilumab on the ciliary beating efficiency using the latter, all analyses were performed at controlled room temperature (20°C–25°C) in vitro in the same primary culture of HNEC with the same conditions as previously described in the methods. Therefore, microbead velocity was used as a marker of the flow generated by the ciliary beating to evaluate the shear stress induced by cilia on the fluid (mean ciliary efficiency index [in mPa]), as previously reported³⁴,³⁶,³⁷ (Supporting Information).

## 2.9 | Wound repair rate in HNEC (time-lapse images)

The in vitro wound repair assay was carried out using a model of mechanical injury adapted from a previously described method performed on fully differentiated cultures of HNEC.³⁸ The repair mechanism was evaluated under the same conditions as previously described in the methods. Supporting Information are also available.

## 2.10 | Statistical analysis

Data are expressed as mean ± standard error of the mean (SEM). SEM was used instead of standard deviation as an estimate of variability among observations because of the size of the samples. Comparisons on tissue samples were made using the Mann-Whitney test. Comparisons of TJ expression, TEER, ciliary beating efficiency, and WRR between cytokine stimulated conditions and basal medium as controls on HNEC were performed using the Wilcoxon test. The latter allows to compare two measurements of a quantitative variable for unpaired data. It is an alternative to the Student's *t*-test when its hypotheses are not valid. A *p*-value ≤0.05 was considered as significant. The statistical

analyses were performed using the R software (v. 4.1.2, R Foundation for Statistical Computing, www.r-project.org).

# 3 | RESULTS

## 3.1 | IL-4 and IL-13 were overexpressed and occludin protein expression was decreased in nasal polyps

IL-4 and IL-13 mRNA were overexpressed in nasal polyps ($n = 9$) when compared to control mucosa ($n = 9$; *p* = 0.0006 and 0.0002, respectively; Figure S1A,B). The protein expression of occludin was decreased in nasal polyps ($n = 5$) when compared to control mucosa ($n = 5$; *p* = 0.0159; Figure S1C).

## 3.2 | IL-4 decreased occludin protein expression in HNEC and dupilumab counteracted this effect

First, we confirmed the mRNA expression of the IL-4Rα in HNEC when compared to negative control (leukemia inhibitory factor receptor, Figure S2). In IL-4 stimulated HNEC, occludin expression was significantly decreased for the 100 ng/mL concentration of IL-4 when compared to control (*p* = 0.0079; Figure 1). No significant changes were observed with IL-13. Dupilumab counteracted the decreased expression of occludin in IL-4 stimulated HNEC whereas anti-IL-6 had no effect (Figure 1). ALI cultures of HNEC stimulated on day 21 with IL-4 1, 10, and 100 ng/mL for 48 h showed a decrease in ZO-1 labeling (Figure 2A–D), whereas no changes were observed with IL-13 (Figure S3). The effects of IL-4 were specifically counteracted by dupilumab (Figure 2F–H).

## 3.3 | IL-4 but not IL-13 decreased TEER in HNEC

The mean ± SEM baseline TEER measurement across all culture wells used for the analysis was 411 ± 144 Ω/cm² ($n = 8$, 64 wells; Figure 3A) prior to cytokine exposure (16 wells corresponding to $n = 2$ patients were discarded due to TEER < 200 Ω/cm²). The control well experiments showed a mild decrease in TEER over the 48 h of cytokine exposure (92.7% of baseline values, 381 ± 112 Ω/cm² at 48 h; Figure 3A). After 48 h of cytokine exposure, only IL-4 (1 ng/mL) exposure revealed a significant decrease in TEER (73.4% of baseline values, 343 ± 89 Ω/cm² at 48 h vs. 467 ± 130 Ω/cm² at baseline, *p* = 0.05; Figure 3A). No significant difference was observed at 48 h for IL-13 at all concentrations tested when compared with unstimulated
an indicator of in vitro permeability.³³ TEER was measured using an epithelial volt ohm meter with an ENDOHM-12 electrode (World Precision Instruments) as described previously.³⁴ Only HNEC with a TEER > 200 Ω/cm² were used (Supporting Information).

## 2.8 | Ciliary beating efficiency in HNEC (high-speed video microscopy)

The effect of IL-4 and IL-13 on the ciliary beat frequency (CBF) of respiratory epithelium has already been described and has been shown to contribute to the impaired mucociliary clearance in vitro.³⁵ Although, CBF is not the best tool to evaluate ciliary beating efficiency, another one described by our team, the efficiency index of ciliary clearance was chosen for the present study.³⁶,³⁷ In order to evaluate further the effects of IL-4, IL-13, and dupilumab on the ciliary beating efficiency using the latter, all analyses were performed at controlled room temperature (20°C–25°C) in vitro in the same primary culture of HNEC with the same conditions as previously described in the methods. Therefore, microbead velocity was used as a marker of the flow generated by the ciliary beating to evaluate the shear stress induced by cilia on the fluid (mean ciliary efficiency index [in mPa]), as previously reported³⁴,³⁶,³⁷ (Supporting Information).

## 2.9 | Wound repair rate in HNEC (time-lapse images)

The in vitro wound repair assay was carried out using a model of mechanical injury adapted from a previously described method performed on fully differentiated cultures of HNEC.³⁸ The repair mechanism was evaluated under the same conditions as previously described in the methods. Supporting Information are also available.

## 2.10 | Statistical analysis

Data are expressed as mean ± standard error of the mean (SEM). SEM was used instead of standard deviation as an estimate of variability among observations because of the size of the samples. Comparisons on tissue samples were made using the Mann-Whitney test. Comparisons of TJ expression, TEER, ciliary beating efficiency, and WRR between cytokine stimulated conditions and basal medium as controls on HNEC were performed using the Wilcoxon test. The latter allows to compare two measurements of a quantitative variable for unpaired data. It is an alternative to the Student's *t*-test when its hypotheses are not valid. A *p*-value ≤0.05 was considered as significant. The statistical

analyses were performed using the R software (v. 4.1.2, R Foundation for Statistical Computing, www.r-project.org).

# 3 | RESULTS

## 3.1 | IL-4 and IL-13 were overexpressed and occludin protein expression was decreased in nasal polyps

IL-4 and IL-13 mRNA were overexpressed in nasal polyps ($n = 9$) when compared to control mucosa ($n = 9$; *p* = 0.0006 and 0.0002, respectively; Figure S1A,B). The protein expression of occludin was decreased in nasal polyps ($n = 5$) when compared to control mucosa ($n = 5$; *p* = 0.0159; Figure S1C).

## 3.2 | IL-4 decreased occludin protein expression in HNEC and dupilumab counteracted this effect

First, we confirmed the mRNA expression of the IL-4Rα in HNEC when compared to negative control (leukemia inhibitory factor receptor, Figure S2). In IL-4 stimulated HNEC, occludin expression was significantly decreased for the 100 ng/mL concentration of IL-4 when compared to control (*p* = 0.0079; Figure 1). No significant changes were observed with IL-13. Dupilumab counteracted the decreased expression of occludin in IL-4 stimulated HNEC whereas anti-IL-6 had no effect (Figure 1). ALI cultures of HNEC stimulated on day 21 with IL-4 1, 10, and 100 ng/mL for 48 h showed a decrease in ZO-1 labeling (Figure 2A–D), whereas no changes were observed with IL-13 (Figure S3). The effects of IL-4 were specifically counteracted by dupilumab (Figure 2F–H).

## 3.3 | IL-4 but not IL-13 decreased TEER in HNEC

The mean ± SEM baseline TEER measurement across all culture wells used for the analysis was 411 ± 144 Ω/cm² ($n = 8$, 64 wells; Figure 3A) prior to cytokine exposure (16 wells corresponding to $n = 2$ patients were discarded due to TEER < 200 Ω/cm²). The control well experiments showed a mild decrease in TEER over the 48 h of cytokine exposure (92.7% of baseline values, 381 ± 112 Ω/cm² at 48 h; Figure 3A). After 48 h of cytokine exposure, only IL-4 (1 ng/mL) exposure revealed a significant decrease in TEER (73.4% of baseline values, 343 ± 89 Ω/cm² at 48 h vs. 467 ± 130 Ω/cm² at baseline, *p* = 0.05; Figure 3A). No significant difference was observed at 48 h for IL-13 at all concentrations tested when compared with unstimulated
<figure><img src="image_2.png" /><figcaption>FIGURE 1 Interleukin (IL)-4 reduced the expression of occludin in human nasal epithelial cells (HNEC) and dupilumab counteracted this effect. Occludin (n = 5) relative protein expression was analyzed by Western blotting using glyceraldehyde 3 phosphate dehydrogenase (GAPDH) as House Keeping Gene (HKG) to normalize protein expression. The y-axis represents the relative expression normalized to HKG. Comparisons were made between groups. **p = 0.0079. Statistical analyses were perfomed using Wilcoxon test. Error bars correspond to the standard error of the mean.</figcaption></figure>

controls (p > 0.05; Figure 3A). Experiments were pursued as described in Section 2 using the following conditions: dupilumab alone, anti-IL-6 alone, IL-4 alone, IL-4 with dupilumab, and IL-4 with anti-IL-6 (Figure 3B). The baseline TEER measurement across all culture wells used for the analysis was 448 ± 123 Ω/cm² (n = 6, 72 wells; Figure 3B) prior to cytokine exposure. After 48 h of cytokine exposure, only IL-4 (1 ng/mL) exposure demonstrated a significant decrease in TEER (77.5% of baseline values, 379 ± 85 Ω/cm² at 48 h vs. 489 ± 65 Ω/cm² at baseline, p = 0.041; Figure 3B). In the presence of IL-4 with a mAb (dupilumab and anti-IL-6), no decrease in TEER was observed at all concentrations tested.

## 3.4 | IL-4 decreased the ciliary beating efficiency in HNEC and dupilumab counteracted this effect

A significant decrease was observed with 1, 10, and 100 ng/mL of IL-4 exposure in comparison to control wells

(n = 6, 84 wells, 0.264 ± 0.09 mPa, 0.216 ± 0.08 mPa, and 0.317 ± 0.11 mPa vs. 0.461 ± 0.16 mPa, p = 0.03, 0.012, and 0.04, respectively; Figure 4A). No significant change was observed at 48 h for IL-13 at all concentrations tested when compared with unstimulated controls (Figure 4B). Experiments were pursued as described in Section 2 using the following conditions: dupilumab alone, anti-IL-6 alone, IL-4 alone, IL-4 with dupilumab, and IL-4 with anti-IL-6 (n = 6, 72 wells; Figure 4C). The mean ciliary index was 0.542 ± 0.24 mPa, 0.557 ± 0.20 mPa, and 0.501 ± 0.72 mPA, for control, dupilumab alone, and anti-IL-6 alone, respectively. IL-4 stimulation significantly reduced the mean ciliary efficiency index at all concentrations tested when compared with the unstimulated control (solid line, Figure 4C). Dupilumab significantly counteracted the effect of IL-4 on ciliary beating for each concentration tested and anti-IL-6 had no effect (dotted line, Figure 4C).

## 3.5 | IL-4 decreased the wound repair rate in HNEC and dupilumab counteracted this effect

As shown in Figure 5A, IL-4 seemed to decrease the WRR from 18 h post-stimulation onwards while no significant effect on the WRR was observed with IL-13 (n = 3, 24 wells, no statistical analysis; Figure 5A,B). Experiments were pursued as described in Section 2 using the following conditions: dupilumab alone, anti-IL-6 alone, IL-4 alone, IL-4 with dupilumab, and IL-4 with anti-IL-6 (n = 6, 72 wells; Figure 5C). In each condition tested, the initial wound areas were similar (213 ± 51 pixels for unstimulated controls vs. 220 ± 55 pixels for IL-4 stimulated wells, p = 0.493; vs. 206 ± 73 pixels for dupilumab stimulated wells, p = 0.889; and vs. 211 ± 37 pixels for anti-IL-6 stimulated wells, p = 0.974). No effect on the WRR was observed with IL-4 (1 ng/mL) (Figure 5D). The stimulation of HNEC with IL-4 (10 ng/mL) decreased the WRR at 42 and 48 h (Figure 5E). A decrease in the WRR was also observed with IL-4 (100 ng/mL) at 24, 42, and 48 h (Figure 5F). The decrease in WRR observed after stimulation by IL-4 (10 and 100 ng/mL) was normalized by dupilumab (Figure 5E,F). The WRR of the 100 ng/mL IL-4 with dupilumab stimulated group was significantly higher than the IL-4 (100 ng/mL) stimulated group (68.3 ± 17.3% vs. 46.9 ± 18.4% at 24 h, p = 0.022; 96.3 ± 6.4% vs. 76.7 ± 18.9% at 42 h, p = 0.006; 99.0 ± 2.4% vs. 85.7 ± 17.0% at 48 h, p = 0.010, respectively; Figure 5F). Dupilumab alone and anti-IL-6 alone had no effect on WRR (Figure 5D-F).
<figure><img src="image_3.png" /><figcaption>FIGURE 1 Interleukin (IL)-4 reduced the expression of occludin in human nasal epithelial cells (HNEC) and dupilumab counteracted this effect. Occludin (n = 5) relative protein expression was analyzed by Western blotting using glyceraldehyde 3 phosphate dehydrogenase (GAPDH) as House Keeping Gene (HKG) to normalize protein expression. The y-axis represents the relative expression normalized to HKG. Comparisons were made between groups. **p = 0.0079. Statistical analyses were perfomed using Wilcoxon test. Error bars correspond to the standard error of the mean.</figcaption></figure>

controls (p > 0.05; Figure 3A). Experiments were pursued as described in Section 2 using the following conditions: dupilumab alone, anti-IL-6 alone, IL-4 alone, IL-4 with dupilumab, and IL-4 with anti-IL-6 (Figure 3B). The baseline TEER measurement across all culture wells used for the analysis was 448 ± 123 Ω/cm² (n = 6, 72 wells; Figure 3B) prior to cytokine exposure. After 48 h of cytokine exposure, only IL-4 (1 ng/mL) exposure demonstrated a significant decrease in TEER (77.5% of baseline values, 379 ± 85 Ω/cm² at 48 h vs. 489 ± 65 Ω/cm² at baseline, *p* = 0.041; Figure 3B). In the presence of IL-4 with a mAb (dupilumab and anti-IL-6), no decrease in TEER was observed at all concentrations tested.

## 3.4 | IL-4 decreased the ciliary beating efficiency in HNEC and dupilumab counteracted this effect

A significant decrease was observed with 1, 10, and 100 ng/mL of IL-4 exposure in comparison to control wells

(n = 6, 84 wells, 0.264 ± 0.09 mPa, 0.216 ± 0.08 mPa, and 0.317 ± 0.11 mPa vs. 0.461 ± 0.16 mPa, *p* = 0.03, 0.012, and 0.04, respectively; Figure 4A). No significant change was observed at 48 h for IL-13 at all concentrations tested when compared with unstimulated controls (Figure 4B). Experiments were pursued as described in Section 2 using the following conditions: dupilumab alone, anti-IL-6 alone, IL-4 alone, IL-4 with dupilumab, and IL-4 with anti-IL-6 (n = 6, 72 wells; Figure 4C). The mean ciliary index was 0.542 ± 0.24 mPa, 0.557 ± 0.20 mPa, and 0.501 ± 0.72 mPA, for control, dupilumab alone, and anti-IL-6 alone, respectively. IL-4 stimulation significantly reduced the mean ciliary efficiency index at all concentrations tested when compared with the unstimulated control (solid line, Figure 4C). Dupilumab significantly counteracted the effect of IL-4 on ciliary beating for each concentration tested and anti-IL-6 had no effect (dotted line, Figure 4C).

## 3.5 | IL-4 decreased the wound repair rate in HNEC and dupilumab counteracted this effect

As shown in Figure 5A, IL-4 seemed to decrease the WRR from 18 h post-stimulation onwards while no significant effect on the WRR was observed with IL-13 (n = 3, 24 wells, no statistical analysis; Figure 5A,B). Experiments were pursued as described in Section 2 using the following conditions: dupilumab alone, anti-IL-6 alone, IL-4 alone, IL-4 with dupilumab, and IL-4 with anti-IL-6 (n = 6, 72 wells; Figure 5C). In each condition tested, the initial wound areas were similar (213 ± 51 pixels for unstimulated controls vs. 220 ± 55 pixels for IL-4 stimulated wells, *p* = 0.493; vs. 206 ± 73 pixels for dupilumab stimulated wells, *p* = 0.889; and vs. 211 ± 37 pixels for anti-IL-6 stimulated wells, *p* = 0.974). No effect on the WRR was observed with IL-4 (1 ng/mL) (Figure 5D). The stimulation of HNEC with IL-4 (10 ng/mL) decreased the WRR at 42 and 48 h (Figure 5E). A decrease in the WRR was also observed with IL-4 (100 ng/mL) at 24, 42, and 48 h (Figure 5F). The decrease in WRR observed after stimulation by IL-4 (10 and 100 ng/mL) was normalized by dupilumab (Figure 5E,F). The WRR of the 100 ng/mL IL-4 with dupilumab stimulated group was significantly higher than the IL-4 (100 ng/mL) stimulated group (68.3 ± 17.3% vs. 46.9 ± 18.4% at 24 h, *p* = 0.022; 96.3 ± 6.4% vs. 76.7 ± 18.9% at 42 h, *p* = 0.006; 99.0 ± 2.4% vs. 85.7 ± 17.0% at 48 h, *p* = 0.010, respectively; Figure 5F). Dupilumab alone and anti-IL-6 alone had no effect on WRR (Figure 5D–F).
<figure><img src="image_3.png" /><figcaption>FIGURE 2 Interleukin (IL)-4 reduced the expression of zonula-occludens (ZO-1) in human nasal epithelial cells (HNEC) and dupilumab counteracted this effect. In order to test the effect of IL-4, and dupilumab on tight junction (TJ), various conditions were tested: IL-4 alone, dupilumab alone, IL-4 with dupilumab, anti-IL-6 alone, and IL-4 with anti-IL-6. Air-liquid interface (ALI) cultures of HNEC were stimulated on day 21 with various concentration of IL-4 (1, 10, and 100 ng/mL) during 48 h (n = 8). ZO-1 (red) expression after immunolabeling, actin fiber labeling (green), and nuclear 4',6-diamidino-2-phénylindole (DAPI) labeling (blue) are shown without any stimulation, control well (A), after stimulation with IL-4 alone at 1 ng/mL (B), 10 ng/mL (C), and 100 ng/mL (D), after stimulation with dupilumab alone (E), and dupilumab with IL-4 at 1 ng/mL (F), 10 ng/mL (G), and 100 ng/mL (H), and after stimulation with anti-IL-6 alone (I), and anti-IL-6 with IL-4 at 1 ng/mL (J), 10 ng/mL (K), and 100 ng/mL (L). To focus on the TJ belt, observations were made at the apical pole of the cell, DAPI and actin labeling cannot be quantified. The analysis was performed on ZO-1 protein expression, which is one of TJ protein complex.</figcaption></figure>

4 | DISCUSSION

The present results show that IL-4 but not IL-13 had a
significant effect on different epithelial functions: IL-4
decreased ZO-1 and occludin expression, TEER, ciliary
beating efficiency, and WRR. Dupilumab alone had no
effect on HNEC but specifically counteracted the effect of
IL-4 on all epithelial functions tested supporting our main
hypothesis.

In the present study, the three increasing concentrations
of IL-4 and IL-13 used to analyze the dose-response effect
were chosen based on previous in vitro studies using
other cytokines.<sup>11,34,39</sup> Conversely to the reported decrease

in CBF and impairment in lateral cell contacts induced
by IL-13,<sup>16</sup> no effect was observed herein. As such, and
based on the results from Wise et al. who reported no
effect of IL-13 on TEER,<sup>23</sup> we thus chose IL-4 as the
working cytokine to test the effect of dupilumab in HNEC.
Although IL-4 and IL-13 both belong to the same family of
cytokines sharing the IL-4Rα subunit, the present study
highlighted their different functional properties.<sup>12</sup> These
may be due to the specific and complex interactions of
each cytokine with their common receptor<sup>12</sup> but also
to the multiple and different signaling pathways that
are activated following their interaction with the other
receptor subunit.<sup>40</sup> In vivo, IL-4 is known to be a regulator
<figure><img src="image_4.png" /><figcaption>FIGURE 2 Interleukin (IL)-4 reduced the expression of zonula-occludens (ZO-1) in human nasal epithelial cells (HNEC) and dupilumab counteracted this effect. In order to test the effect of IL-4, and dupilumab on tight junction (TJ), various conditions were tested: IL-4 alone, dupilumab alone, IL-4 with dupilumab, anti-IL-6 alone, and IL-4 with anti-IL-6. Air-liquid interface (ALI) cultures of HNEC were stimulated on day 21 with various concentration of IL-4 (1, 10, and 100 ng/mL) during 48 h (n = 8). ZO-1 (red) expression after immunolabeling, actin fiber labeling (green), and nuclear 4',6-diamidino-2-phénylindole (DAPI) labeling (blue) are shown without any stimulation, control well (A), after stimulation with IL-4 alone at 1 ng/mL (B), 10 ng/mL (C), and 100 ng/mL (D), after stimulation with dupilumab alone (E), and dupilumab with IL-4 at 1 ng/mL (F), 10 ng/mL (G), and 100 ng/mL (H), and after stimulation with anti-IL-6 alone (I), and anti-IL-6 with IL-4 at 1 ng/mL (J), 10 ng/mL (K), and 100 ng/mL (L). To focus on the TJ belt, observations were made at the apical pole of the cell, DAPI and actin labeling cannot be quantified. The analysis was performed on ZO-1 protein expression, which is one of TJ protein complex.</figcaption></figure>

4 | DISCUSSION

The present results show that IL-4 but not IL-13 had a
significant effect on different epithelial functions: IL-4
decreased ZO-1 and occludin expression, TEER, ciliary
beating efficiency, and WRR. Dupilumab alone had no
effect on HNEC but specifically counteracted the effect of
IL-4 on all epithelial functions tested supporting our main
hypothesis.

In the present study, the three increasing concentrations
of IL-4 and IL-13 used to analyze the dose-response effect
were chosen based on previous in vitro studies using
other cytokines.<sup>11,34,39</sup> Conversely to the reported decrease

in CBF and impairment in lateral cell contacts induced
by IL-13,<sup>16</sup> no effect was observed herein. As such, and
based on the results from Wise et al. who reported no
effect of IL-13 on TEER,<sup>23</sup> we thus chose IL-4 as the
working cytokine to test the effect of dupilumab in HNEC.
Although IL-4 and IL-13 both belong to the same family of
cytokines sharing the IL-4Rα subunit, the present study
highlighted their different functional properties.<sup>12</sup> These
may be due to the specific and complex interactions of
each cytokine with their common receptor<sup>12</sup> but also
to the multiple and different signaling pathways that
are activated following their interaction with the other
receptor subunit.<sup>40</sup> In vivo, IL-4 is known to be a regulator
<figure><img src="image_4.png" /><figcaption>FIG URE 3 Interleukin (IL)-4 but not IL-13 decreased transepithelial electrical resistance (TEER) in human nasal epithelial cells (HNEC). In order to test the effect of IL-4, IL-13, and dupilumab on TEER, various conditions were tested: IL-4 alone, IL-13 alone, dupilumab alone, IL-4 with dupilumab, anti-IL-6 alone, and IL-4 with anti-IL-6. Air-liquid interface (ALI) cultures of HNEC were stimulated on day 21 with various concentrations of IL-4 or IL-13 (1, 10, and 100 ng/mL) during 48 h and compared to unstimulated cultures. TEER measurements (expressed in Ω/cm²) were performed in unstimulated controls (black), IL-4 alone at 1 ng/mL (light blue), 10 ng/mL (blue), and 100 ng/mL (dark blue), IL-13 alone at 1 ng/mL (light orange), 10 ng/mL (orange), and 100 ng/mL (dark orange), IL-4 with dupilumab (light purple, purple, and dark purple), and IL-4 with anti-IL-6 (light green, green, and dark green). IL-4 at 1 ng/mL exposure demonstrated a significant decrease in TEER, whereas IL-13 had no effect (n = 8). Effect of IL-4 was counteracted by dupilumab and anti-IL6 (n = 6). (A) *p* = 0.05 and (B) *p* = 0.041. Statistical analyses were performed using Wilcoxon test. Error bars correspond to the standard error of the mean.</figcaption></figure>

of T2 lymphocyte differentiation and class switching of B cells (IgG1 to IgE),⁴¹ which is in line with its direct in vitro effect on several epithelial functions reported herein.

CRSwNP is the result of a chronic nasal inflammatory response triggered by airway epithelial disruption regardless of the initial environmental insult.²,⁵,¹¹,⁴²,⁴³ This insult could increase cytokine expression in nasal polyps.¹⁰ The TJ proteins, including occludin and ZO-1, are crucial in

ensuring the efficacy of the epithelial barrier function.⁵,⁴⁴ Both IL-4 and IL-13 are supposed to decrease the expression of TJ proteins in vitro²³ and therefore, contribute to the alteration of the epithelial barrier described in CRSwNP.²³,²⁴ However, we only observed this under IL-4 stimulation, in accordance with Wise et al.²³ Similarly, a decrease in TEER was observed with IL-4 but not IL-13. Then, mucociliary clearance, which represents a key
<figure><img src="image_5.png" /><figcaption>**FIG URE 3** Interleukin (IL)-4 but not IL-13 decreased transepithelial electrical resistance (TEER) in human nasal epithelial cells (HNEC). In order to test the effect of IL-4, IL-13, and dupilumab on TEER, various conditions were tested: IL-4 alone, IL-13 alone, dupilumab alone, IL-4 with dupilumab, anti-IL-6 alone, and IL-4 with anti-IL-6. Air-liquid interface (ALI) cultures of HNEC were stimulated on day 21 with various concentrations of IL-4 or IL-13 (1, 10, and 100 ng/mL) during 48 h and compared to unstimulated cultures. TEER measurements (expressed in Ω/cm²) were performed in unstimulated controls (black), IL-4 alone at 1 ng/mL (light blue), 10 ng/mL (blue), and 100 ng/mL (dark blue), IL-13 alone at 1 ng/mL (light orange), 10 ng/mL (orange), and 100 ng/mL (dark orange), IL-4 with dupilumab (light purple, purple, and dark purple), and IL-4 with anti-IL-6 (light green, green, and dark green). IL-4 at 1 ng/mL exposure demonstrated a significant decrease in TEER, whereas IL-13 had no effect (n = 8). Effect of IL-4 was counteracted by dupilumab and anti-IL6 (n = 6). (A) *p* = 0.05 and (B) *p* = 0.041. Statistical analyses were performed using Wilcoxon test. Error bars correspond to the standard error of the mean.</figcaption></figure>

of T2 lymphocyte differentiation and class switching of B cells (IgG1 to IgE),⁴¹ which is in line with its direct in vitro effect on several epithelial functions reported herein.

CRSwNP is the result of a chronic nasal inflammatory response triggered by airway epithelial disruption regardless of the initial environmental insult.<sup>2,5,11,42,43</sup> This insult could increase cytokine expression in nasal polyps.<sup>10</sup> The TJ proteins, including occludin and ZO-1, are crucial in

ensuring the efficacy of the epithelial barrier function.<sup>5,44</sup> Both IL-4 and IL-13 are supposed to decrease the expression of TJ proteins in vitro<sup>23</sup> and therefore, contribute to the alteration of the epithelial barrier described in CRSwNP.<sup>23,24</sup> However, we only observed this under IL-4 stimulation, in accordance with Wise et al.<sup>23</sup> Similarly, a decrease in TEER was observed with IL-4 but not IL-13. Then, mucociliary clearance, which represents a key
<figure><img src="image_5.png" /><figcaption>FIG URE 4 Interleukin (IL)-4 decreased the ciliary beating efficiency and dupilumab in vitro counteracted this effect. In order to test the effect of IL-4, IL-13, and dupilumab on ciliary beating efficiency, various conditions were tested: IL-4 alone, IL-13 alone, dupilumab alone, IL-4 with dupilumab, anti-IL-6 alone, and IL-4 with anti-IL-6. The ciliary beating efficiency (mPa) was evaluated after 48 h of IL-4 (light blue, blue, and dark blue) (A) and IL-13 (light orange, orange, and dark orange) (B) stimulation at various concentrations (1, 10, and 100 ng/mL) and compared with unstimulated controls (black; *n* = 6). Stimulation of human nasal epithelial cells (HNEC) with IL-4 alone at 1, 10, and 100 ng/mL decreased ciliary beating efficiency (*p* < 0.05). The ciliary beating efficiency (mPa) was then evaluated after 48 h of stimulation with IL-4 alone (light blue, blue, and dark blue), IL-4 with dupilumab (light purple, purple, and dark purple), and IL-4 with anti-IL-6 (light green, green, and dark green) at various concentrations of IL-4 for each group (1, 10, and 100 ng/mL) (C). Comparisons were made with unstimulated controls (black; *n* = 6, *p* = 0.005, **p** = 0.03, and ***p** = 0.007). The effect of IL-4 was counteracted specifically by dupilumab (respectively, *p* = 0.003, **p** = 0.016, and ***p** = 0.003) but not by anti-IL-6. Statistical analyses were performed using Wilcoxon test. Error bars correspond to the standard error of the mean.</figcaption></figure>

step in the defense capacity of the airway epithelium,
could also be altered in CRSwNP.¹⁶,¹⁷,³⁵,⁴⁵ Previous stud-
ies demonstrated that IL-4 and IL-13 both significantly
reduced CBF¹⁷,³⁵ and that IL-13 reduced cell differentia-
tion in HNECs from CRSwNP patients.¹⁷ The hypothesis
is that a decrease in ciliary function could contribute to
the persistence of pathogens at the apical pole of epithelial
cells. In the present study, IL-4 but not IL-13 significantly
reduced the mean ciliary efficiency index when compared
to controls. These results differ from those reported by Jiao
et al. who found an effect of IL-13 on CBF when study-
ing the proportion of ciliated cells,¹⁷ a parameter that is
less specific than the efficiency index for CRSwNP.³⁴,³⁶,³⁷
In another study, the RNA sequencing of sinus epithe-
lial brushings from CRSwNP patients found an increase
in tuft cell transcripts and a decrease in ciliated cells after

IL-13 stimulation, but the ciliary beating efficiency per se
was not studied.²¹ As TJ alterations are directly correlated
with cell migration and epithelial wound healing,⁴⁶⁻⁴⁸ we
expected results similar to those described by Wise et al.,
that is an effect with IL-4 but not IL-13.²³ A link between a
decreased WRR and a decreased ciliary beating efficiency
could be drawn under the hypothesis of a complete loss
of usual function of the epithelium when exposed to an
inflammatory environment, herein characterized by IL-4
exposure.

A defective epithelial barrier with decreased tissue resis-
tance and decreased expression of TJ proteins⁵ is one
of the numerous hypothesis underlying the CRSwNP
pathogenesis.⁴⁹ Therefore, restoring the integrity of the
epithelial barrier becomes a key element in this disease.
In the present study, dupilumab specifically restored all
<figure><img src="image_6.png" /><figcaption>FIGURE 4 Interleukin (IL)-4 decreased the ciliary beating efficiency and dupilumab in vitro counteracted this effect. In order to test the effect of IL-4, IL-13, and dupilumab on ciliary beating efficiency, various conditions were tested: IL-4 alone, IL-13 alone, dupilumab alone, IL-4 with dupilumab, anti-IL-6 alone, and IL-4 with anti-IL-6. The ciliary beating efficiency (mPa) was evaluated after 48 h of IL-4 (light blue, blue, and dark blue) (A) and IL-13 (light orange, orange, and dark orange) (B) stimulation at various concentrations (1, 10, and 100 ng/mL) and compared with unstimulated controls (black; *n* = 6). Stimulation of human nasal epithelial cells (HNEC) with IL-4 alone at 1, 10, and 100 ng/mL decreased ciliary beating efficiency (*p* < 0.05). The ciliary beating efficiency (mPa) was then evaluated after 48 h of stimulation with IL-4 alone (light blue, blue, and dark blue), IL-4 with dupilumab (light purple, purple, and dark purple), and IL-4 with anti-IL-6 (light green, green, and dark green) at various concentrations of IL-4 for each group (1, 10, and 100 ng/mL) (C). Comparisons were made with unstimulated controls (black; *n* = 6, *p* = 0.005, **p** = 0.03, and ***p** = 0.007). The effect of IL-4 was counteracted specifically by dupilumab (respectively, *p* = 0.003, **p** = 0.016, and ***p** = 0.003) but not by anti-IL-6. Statistical analyses were performed using Wilcoxon test. Error bars correspond to the standard error of the mean.</figcaption></figure>

step in the defense capacity of the airway epithelium,
could also be altered in CRSwNP.¹⁶,¹⁷,³⁵,⁴⁵ Previous stud-
ies demonstrated that IL-4 and IL-13 both significantly
reduced CBF¹⁷,³⁵ and that IL-13 reduced cell differentia-
tion in HNECs from CRSwNP patients.¹⁷ The hypothesis
is that a decrease in ciliary function could contribute to
the persistence of pathogens at the apical pole of epithelial
cells. In the present study, IL-4 but not IL-13 significantly
reduced the mean ciliary efficiency index when compared
to controls. These results differ from those reported by Jiao
et al. who found an effect of IL-13 on CBF when study-
ing the proportion of ciliated cells,¹⁷ a parameter that is
less specific than the efficiency index for CRSwNP.³⁴,³⁶,³⁷
In another study, the RNA sequencing of sinus epithe-
lial brushings from CRSwNP patients found an increase
in tuft cell transcripts and a decrease in ciliated cells after

IL-13 stimulation, but the ciliary beating efficiency per se
was not studied.²¹ As TJ alterations are directly correlated
with cell migration and epithelial wound healing,⁴⁶⁻⁴⁸ we
expected results similar to those described by Wise et al.,
that is an effect with IL-4 but not IL-13.²³ A link between a
decreased WRR and a decreased ciliary beating efficiency
could be drawn under the hypothesis of a complete loss
of usual function of the epithelium when exposed to an
inflammatory environment, herein characterized by IL-4
exposure.

A defective epithelial barrier with decreased tissue resis-
tance and decreased expression of TJ proteins⁵ is one
of the numerous hypothesis underlying the CRSwNP
pathogenesis.⁴⁹ Therefore, restoring the integrity of the
epithelial barrier becomes a key element in this disease.
In the present study, dupilumab specifically restored all
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>FIGURE 5 Interleukin (IL)-4 decreased the wound repair rate (WRR) in vitro and dupilumab counteracted this effect. In order to test the effect of IL-4, IL-13, and dupilumab on the repair rate, various conditions were tested: IL-4 alone, IL-13 alone, dupilumab alone, IL-4 with dupilumab, anti-IL-6 alone, and IL-4 with anti-IL-6. The repair rate was evaluated as the proportion of the wounded area closed at each time point for IL-4 and IL-13 stimulated wells (either 1, 10, or 100 ng/mL) and compared to the one of unstimulated control wells, first for IL-4 alone (n = 3) (A) and IL-13 alone (n = 3) (B) to test their effect on human nasal epithelial cells (HNEC) (no statistical analysis performed). Then, experiments were pursued with IL-4 alone, dupilumab alone, anti-IL-6 alone, IL-4 with dupilumab, and IL-4 with anti-IL-6 (n = 5) (C–F). Statistical analyses were perfomed using Wilcoxon test. Error bars correspond to the standard error of the mean. No effect on the WRR was observed with IL-4 alone at 1 ng/mL (D). At 42 and 48 h, the 10 ng/mL IL-4 alone stimulated group had a lower repair rate than the unstimulated control group (76.7 ± 18.9% vs. 99.5 ± 1.4% at 42 h, *p* = 0.001; 88.6 ± 16.9% vs. 100.0 ± 0.0% at 48 h, *p* = 0.013) (E). When HNEC were stimulated with IL-4 at 10 ng/mL with dupilumab, the WRR was normalized (E). At 24, 42, and 48 h, the 100 ng/mL IL-4 stimulated group had a lower repair rate than the unstimulated control group (46.9 ± 18.4% vs. 69.8 ± 23.9%, *p* = 0.037 at 24 h; 76.7 ± 18.9% vs. 99.5 ± 1.4% at 42 h, *p* = 0.001; 85.7 ± 17.0% vs. 100.0 ± 0.0% at 48 h, *p* = 0.001). When HNEC were stimulated with IL-4 at 100 ng/mL with dupilumab, the WRR was normalized and the 100 ng/mL IL-4 with dupilumab stimulated group had a higher repair rate than the 100 ng/mL IL-4 alone stimulated group (68.3 ± 17.3% vs. 46.9 ± 18.4% at 24 h, *p* = 0.022; 96.3 ± 6.4% vs. 76.7 ± 18.9% at 42 h, *p* = 0.006; 99.0 ± 2.4% vs. 85.7 ± 17.0% at 48 h, *p* = 0.010, for IL-4 at 100 ng/mL with dupilumab vs. IL-4 100 ng/mL alone, respectively) (F). \*Statistical significance between control and IL-4 stimulated wells (*p* < 0.05); \*\*between IL-4 and IL-4 with dupilumab stimulated wells.</figcaption></figure>
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /><figcaption>FIG URE 5 Interleukin (IL)-4 decreased the wound repair rate (WRR) in vitro and dupilumab counteracted this effect. In order to test the effect of IL-4, IL-13, and dupilumab on the repair rate, various conditions were tested: IL-4 alone, IL-13 alone, dupilumab alone, IL-4 with dupilumab, anti-IL-6 alone, and IL-4 with anti-IL-6. The repair rate was evaluated as the proportion of the wounded area closed at each time point for IL-4 and IL-13 stimulated wells (either 1, 10, or 100 ng/mL) and compared to the one of unstimulated control wells, first for IL-4 alone (n = 3) (A) and IL-13 alone (n = 3) (B) to test their effect on human nasal epithelial cells (HNEC) (no statistical analysis performed). Then, experiments were pursued with IL-4 alone, dupilumab alone, anti-IL-6 alone, IL-4 with dupilumab, and IL-4 with anti-IL-6 (n = 5) (C–F). Statistical analyses were perfomed using Wilcoxon test. Error bars correspond to the standard error of the mean. No effect on the WRR was observed with IL-4 alone at 1 ng/mL (D). At 42 and 48 h, the 10 ng/mL IL-4 alone stimulated group had a lower repair rate than the unstimulated control group (76.7 ± 18.9% vs. 99.5 ± 1.4% at 42 h, *p* = 0.001; 88.6 ± 16.9% vs. 100.0 ± 0.0% at 48 h, *p* = 0.013) (E). When HNEC were stimulated with IL-4 at 10 ng/mL with dupilumab, the WRR was normalized (E). At 24, 42, and 48 h, the 100 ng/mL IL-4 stimulated group had a lower repair rate than the unstimulated control group (46.9 ± 18.4% vs. 69.8 ± 23.9%, *p* = 0.037 at 24 h; 76.7 ± 18.9% vs. 99.5 ± 1.4% at 42 h, *p* = 0.001; 85.7 ± 17.0% vs. 100.0 ± 0.0% at 48 h, *p* = 0.001). When HNEC were stimulated with IL-4 at 100 ng/mL with dupilumab, the WRR was normalized and the 100 ng/mL IL-4 with dupilumab stimulated group had a higher repair rate than the 100 ng/mL IL-4 alone stimulated group (68.3 ± 17.3% vs. 46.9 ± 18.4% at 24 h, *p* = 0.022; 96.3 ± 6.4% vs. 76.7 ± 18.9% at 42 h, *p* = 0.006; 99.0 ± 2.4% vs. 85.7 ± 17.0% at 48 h, *p* = 0.010, for IL-4 at 100 ng/mL with dupilumab vs. IL-4 100 ng/mL alone, respectively) (F). \*Statistical significance between control and IL-4 stimulated wells (*p* < 0.05); \*\*between IL-4 and IL-4 with dupilumab stimulated wells.</figcaption></figure>
epithelial functions (TJ protein expression, ciliary beating efficiency, and WRR) altered by IL-4; its effect on TEER measurements, however, was not specific. We could suggest that in vitro, a decreased TEER under IL-4 exposure is counteracted in the presence of mAbs, whether they are specific (dupilumab) or not (anti-IL-6), partly due to the sensitivity of HNEC to their environment.

Among the limitations of the present study, the lack of data regarding goblet cell secretions under IL-13 stimulations could be raised; the latter however has already been well described in the literature.¹⁶ Another limitation could be the lack of nice dose response curves with IL-4 stimulation, which was unexpected. Indeed, the concentrations chosen were close to the one found in vivo, but the response is complex, keeping in mind that cytokines have paracrine effects. In addition, IL-4 was tested alone and a synergistic effect with other cytokines should be considered. However, since no effect of IL-13 was identified, we decided not to test the epithelial functions under the co-exposition of IL-4 and IL-13. Furthermore, addressing the embryological distinction between septal and ethmoidal mucosae is conceivable, but unlikely to significantly impact our results. The common use of septal mucosae or inferior turbinate biopsies as controls in unraveling CRSwNP pathogenesis reinforces this assertion.⁵⁰⁻⁵⁵ Finally, our choice to focus on CRSwNP patients could be a limitation, but since dupilumab is only authorized in these patients, it did not appear relevant to study patients with CRSsNP or allergic rhinitis.

## 5 | CONCLUSION

In conclusion, dupilumab, in vitro, maintained epithelial integrity by counteracting the effects of IL-4 on the epithelial barrier (increased epithelial permeability, decreased ciliary beating efficiency, and decreased WRR). These findings suggest that the efficacy of dupilumab is underlined by its capacity to prevent the alteration of epithelial functions, one of the key features of CRSwNP pathogenesis.

## AUTHOR CONTRIBUTIONS

**Conceptualization:** Emilie Bequignon, Laure Favot, Maxime Fieux, Stéphane Tringali, André Coste, Jean Claude Lecron, Florent Carsuzaa, and Yvan Bellanger.
**Data curation:** Maxime Fieux, Florent Carsuzaa, and Yvan Bellanger.
**Formal analysis:** Emilie Bequignon, Maxime Fieux, Laure Favot, Sophie Bartier, Xavier Dufour, Matthieu Bainaud, Bruno Louis, Florent Carsuzaa, and Yvan Bellanger.
**Methodology:** André Coste, Emilie Bequignon, Maxime Fieux, Florent Carsuzaa, and Yvan Bellanger.
**Project administration:** André Coste, Bruno Louis, Emilie Bequignon, and Laure Favot.
**Resources:** Bruno Louis and Laure Favot.
**Software:** Maxime Fieux and Bruno Louis.
**Supervision:** Bruno Louis, Laure Favot, Emilie Bequignon, Stéphane Tringali, Xavier Dufour, Jean Claude Lecron, and André Coste.
**Validation:** Emilie Bequignon and Laure Favot.
**Visualization:** André Coste, Xavier Dufour, and Stéphane Tringali.
**Writing—original draft:** Maxime Fieux, Florent Carsuzaa, Yvan Bellanger, Emilie Bequignon, Jean Claude Lecron, and Laure Favot.
**Writing—review and editing:** Emilie Bequignon, Laure Favot, André Coste, Jean Claude Lecron, Maxime Fieux, Stéphane Tringali, Xavier Dufour, Matthieu Bainaud, Florent Carsuzaa, and Yvan Bellanger. All the authors have read and agreed to the published version of the manuscript.

## ACKNOWLEDGMENTS

The authors are thankful to all members of the lab in Poitiers and Créteil. We also thank Véréna Landel (Direction de le Recherche en Santé, Hospices Civils de Lyon) for her help in manuscript preparation. This work was partly supported by a grant from the Société Française d'Oto-Rhino-Laryngologie et de Chirurgie Cervico-Faciale (SFORL), the Collège Français d'Oto-Rhino-Laryngologie et de Chirurgie Cervico-Faciale (CFORL&CCF), and Amplifon.

## CONFLICT OF INTEREST STATEMENT

M.F., E.B., F.C., X.D., and A.C. have received honoraria from Sanofi and GlaxoSmith. Remaining authors declare they have no conflicts of interest in relation to this work.

## DATA AVAILABILITY STATEMENT

Data supporting reported results can be found in the Laboratoire Inflammation Tissus Epithéliaux et Cytokines (UR15560), Université de Poitiers (86000 Poitiers), and the Laboratoire de l'Institut Mondor de Recherche Biomédicale (INSERM U955, CNRS EMR 7000, 93000 Créteil).

## ETHICS STATEMENT

The study was approved by the Institutional Ethics Committee on Human Experimentation of the University Hospital of Poitiers and received the agreement of the French “Ministère de l’Enseignement Supérieur et de la Recherche” (DC-20193746, December 2019). Regarding primary culture of human nasal epithelial cells, all the patients had given informed consent, and the study was approved by the local ethics committee (CPP IDF X 2016-01-01).

## CONSENT FOR PUBLICATION

Informed consent was obtained from all subjects involved in the study.
epithelial functions (TJ protein expression, ciliary beating efficiency, and WRR) altered by IL-4; its effect on TEER measurements, however, was not specific. We could suggest that in vitro, a decreased TEER under IL-4 exposure is counteracted in the presence of mAbs, whether they are specific (dupilumab) or not (anti-IL-6), partly due to the sensitivity of HNEC to their environment.

Among the limitations of the present study, the lack of data regarding goblet cell secretions under IL-13 stimulations could be raised; the latter however has already been well described in the literature.¹⁶ Another limitation could be the lack of nice dose response curves with IL-4 stimulation, which was unexpected. Indeed, the concentrations chosen were close to the one found in vivo, but the response is complex, keeping in mind that cytokines have paracrine effects. In addition, IL-4 was tested alone and a synergistic effect with other cytokines should be considered. However, since no effect of IL-13 was identified, we decided not to test the epithelial functions under the co-exposition of IL-4 and IL-13. Furthermore, addressing the embryological distinction between septal and ethmoidal mucosae is conceivable, but unlikely to significantly impact our results. The common use of septal mucosae or inferior turbinate biopsies as controls in unraveling CRSwNP pathogenesis reinforces this assertion.⁵⁰⁻⁵⁵ Finally, our choice to focus on CRSwNP patients could be a limitation, but since dupilumab is only authorized in these patients, it did not appear relevant to study patients with CRSsNP or allergic rhinitis.

## 5 | CONCLUSION

In conclusion, dupilumab, in vitro, maintained epithelial integrity by counteracting the effects of IL-4 on the epithelial barrier (increased epithelial permeability, decreased ciliary beating efficiency, and decreased WRR). These findings suggest that the efficacy of dupilumab is underlined by its capacity to prevent the alteration of epithelial functions, one of the key features of CRSwNP pathogenesis.

## AUTHOR CONTRIBUTIONS

**Conceptualization:** Emilie Bequignon, Laure Favot, Maxime Fieux, Stéphane Tringali, André Coste, Jean Claude Lecron, Florent Carsuzaa, and Yvan Bellanger.
**Data curation:** Maxime Fieux, Florent Carsuzaa, and Yvan Bellanger.
**Formal analysis:** Emilie Bequignon, Maxime Fieux, Laure Favot, Sophie Bartier, Xavier Dufour, Matthieu Bainaud, Bruno Louis, Florent Carsuzaa, and Yvan Bellanger.
**Methodology:** André Coste, Emilie Bequignon, Maxime Fieux, Florent Carsuzaa, and Yvan Bellanger.
**Project administration:** André Coste, Bruno Louis, Emilie Bequignon, and Laure Favot.
**Resources:** Bruno Louis and Laure Favot.
**Software:** Maxime Fieux and Bruno Louis.
**Supervision:** Bruno Louis, Laure Favot, Emilie Bequignon, Stéphane Tringali, Xavier Dufour, Jean Claude Lecron, and André Coste.
**Validation:** Emilie Bequignon and Laure Favot.
**Visualization:** André Coste, Xavier Dufour, and Stéphane Tringali.
**Writing—original draft:** Maxime Fieux, Florent Carsuzaa, Yvan Bellanger, Emilie Bequignon, Jean Claude Lecron, and Laure Favot.
**Writing—review and editing:** Emilie Bequignon, Laure Favot, André Coste, Jean Claude Lecron, Maxime Fieux, Stéphane Tringali, Xavier Dufour, Matthieu Bainaud, Florent Carsuzaa, and Yvan Bellanger. All the authors have read and agreed to the published version of the manuscript.

## ACKNOWLEDGMENTS

The authors are thankful to all members of the lab in Poitiers and Créteil. We also thank Véréna Landel (Direction de le Recherche en Santé, Hospices Civils de Lyon) for her help in manuscript preparation. This work was partly supported by a grant from the Société Française d'Oto-Rhino-Laryngologie et de Chirurgie Cervico-Faciale (SFORL), the Collège Français d'Oto-Rhino-Laryngologie et de Chirurgie Cervico-Faciale (CFORL&CCF), and Amplifon.

## CONFLICT OF INTEREST STATEMENT

M.F., E.B., F.C., X.D., and A.C. have received honoraria from Sanofi and GlaxoSmith. Remaining authors declare they have no conflicts of interest in relation to this work.

## DATA AVAILABILITY STATEMENT

Data supporting reported results can be found in the Laboratoire Inflammation Tissus Epithéliaux et Cytokines (UR15560), Université de Poitiers (86000 Poitiers), and the Laboratoire de l'Institut Mondor de Recherche Biomédicale (INSERM U955, CNRS EMR 7000, 93000 Créteil).

## ETHICS STATEMENT

The study was approved by the Institutional Ethics Committee on Human Experimentation of the University Hospital of Poitiers and received the agreement of the French "Ministère de l'Enseignement Supérieur et de la Recherche" (DC-20193746, December 2019). Regarding primary culture of human nasal epithelial cells, all the patients had given informed consent, and the study was approved by the local ethics committee (CPP IDF X 2016-01-01).

## CONSENT FOR PUBLICATION

Informed consent was obtained from all subjects involved in the study.
# DATA, MATERIALS, AND/OR CODE AVAILABILITY

No additional data are available.

## ORCID

Maxime Fieux MD, PhD https://orcid.org/0000-0001-8317-2286

Florent Carsuzaa MD https://orcid.org/0000-0001-5574-8559

Sophie Bartier MD https://orcid.org/0000-0002-5817-691X

Bruno Louis PhD https://orcid.org/0000-0001-9587-9932

## REFERENCES

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2. Fokkens WJ, Lund VJ, Hopkins C, et al. European position paper on rhinosinusitis and nasal polyps 2020. *Rhinology*. 2020;58(suppl S29):1-464. doi:10.4193/Rhin20.600

3. Tomassen P, Vandeplas G, Van Zele T, et al. Inflammatory endotypes of chronic rhinosinusitis based on cluster analysis of biomarkers. *J Allergy Clin Immunol*. 2016;137(5):1449-1456.e4. doi:10.1016/j.jaci.2015.12.1324

4. Laidlaw TM, Mullol J, Woessner KM, Amin N, Mannent LP. Chronic rhinosinusitis with nasal polyps and asthma. *J Allergy Clin Immunol Pract*. 2021;9(3):1133-1141. doi:10.1016/j.jaip.2020.09.063

5. Soyka MB, Wawrzyniak P, Eiwegger T, et al. Defective epithelial barrier in chronic rhinosinusitis: the regulation of tight junctions by IFN-γ and IL-4. *J Allergy Clin Immunol*. 2012;130(5):1087-1096.e10. doi:10.1016/j.jaci.2012.05.052

6. Coste A, Brugel L, Maître B, et al. Inflammatory cells as well as epithelial cells in nasal polyps express vascular endothelial growth factor. *Eur Respir J*. 2000;15(2):367-372. doi:10.1034/j.1399-3003.2000.15b24.x

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10. Carsuzaa F, Béquignon É, Dufour X, de Bonnecaze G, Lecron JC, Favot L. Cytokine signature and involvement in chronic rhinosinusitis with nasal polyps. *Int J Mol Sci*. 2021;23(1):417. doi:10.3390/ijms23010417

11. Bequignon E, Mangin D, Bécaud J, et al. Pathogenesis of chronic rhinosinusitis with nasal polyps: role of IL-6 in airway epithelial cell dysfunction. *J Transl Med*. 2020;18(1):136. doi:10.1186/s12967-020-02309-9

12. LaPorte SL, Juo ZS, Vaclavikova J, et al. Molecular and structural basis of cytokine receptor pleiotropy in the interleukin-4/13 system. *Cell*. 2008;132(2):259-272. doi:10.1016/j.cell.2007.12.030

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22. Lee SN, Ahn JS, Lee SG, Lee HS, Choi AMK, Yoon JH. Integrins αvβ5 and αvβ6 mediate IL-4-induced collective migration in human airway epithelial cells. *Am J Respir Cell Mol Biol*. 2019;60(4):420-433. doi:10.1165/rcmb.2018-0081OC

23. Wise SK, Laury AM, Katz EH, Den Beste KA, Parkos CA, Nusrat A. Interleukin-4 and interleukin-13 compromise the sinonasal epithelial barrier and perturb intercellular junction protein expression. *Int Forum Allergy Rhinol*. 2014;4(5):361-370. doi:10.1002/aler.21298

24. Wise SK, Den Beste KA, Hoddeson EK, Parkos CA, Nusrat A. Sinonasal epithelial wound resealing in an in vitro model: inhibition of wound closure with IL-4 exposure. *Int Forum Allergy Rhinol*. 2013;3(6):439-449. doi:10.1002/aler.21158

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26. Le Floc'h A, Allinne J, Nagashima K, et al. Dual blockade of IL-4 and IL-13 with dupilumab, an IL-4Rα antibody, is required to broadly inhibit type 2 inflammation. *Allergy*. 2020;75(5):1188-1204. doi:10.1111/all.14151
DATA, MATERIALS, AND/OR CODE
AVAILABILITY

No additional data are available.

ORCID

Maxime Fieux MD, PhD https://orcid.org/0000-0001-8317-2286

Florent Carsuzaa MD https://orcid.org/0000-0001-5574-8559

Sophie Bartier MD https://orcid.org/0000-0002-5817-691X

Bruno Louis PhD https://orcid.org/0000-0001-9587-9932

REFERENCES

1. Hastan D, Fokkens WJ, Bachert C, et al. Chronic rhinosinusitis in Europe–an underestimated disease. A GA²LEN study. *Allergy*. 2011;66(9):1216-1223. doi:10.1111/j.1398-9995.2011.02646.x

2. Fokkens WJ, Lund VJ, Hopkins C, et al. European position paper on rhinosinusitis and nasal polyps 2020. *Rhinology*. 2020;58(suppl S29):1-464. doi:10.4193/Rhin20.600

3. Tomassen P, Vandeplas G, Van Zele T, et al. Inflammatory endotypes of chronic rhinosinusitis based on cluster analysis of biomarkers. *J Allergy Clin Immunol*. 2016;137(5):1449-1456.e4. doi:10.1016/j.jaci.2015.12.1324

4. Laidlaw TM, Mullol J, Woessner KM, Amin N, Mannent LP. Chronic rhinosinusitis with nasal polyps and asthma. *J Allergy Clin Immunol Pract*. 2021;9(3):1133-1141. doi:10.1016/j.jaip.2020.09.063

5. Soyka MB, Wawrzyniak P, Eiwegger T, et al. Defective epithelial barrier in chronic rhinosinusitis: the regulation of tight junctions by IFN-γ and IL-4. *J Allergy Clin Immunol*. 2012;130(5):1087-1096.e10. doi:10.1016/j.jaci.2012.05.052

6. Coste A, Brugel L, Maître B, et al. Inflammatory cells as well as epithelial cells in nasal polyps express vascular endothelial growth factor. *Eur Respir J*. 2000;15(2):367-372. doi:10.1034/j.1399-3003.2000.15b24.x

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28. Georas SN, Rezaee F. Epithelial barrier function: at the front line of asthma immunology and allergic airway inflammation. *J Allergy Clin Immunol*. 2014;134(3):509-520. doi:10.1016/j.jaci. 2014.05.049

29. Coste A, Rateau JG, Bernaudin JF, Peynègre R, Escudier E. Nasal polyposis pathogenesis: a flow cytometric and immunohistochemical study of epithelial cell proliferation. *Acta Otolaryngol (Stockh)*. 1996;116(5):755-761. doi:10.3109/00016489609137920

30. Coste A, Rateau JG, Roudot-Thoraval F, et al. Increased epithelial cell proliferation in nasal polyps. *Arch Otolaryngol Head Neck Surg*. 1996;122(4):432-436. doi:10.1001/archotol.1996. 01890160072013

31. Papon JF, Coste A, Gendron MC, et al. HLA-DR and ICAM-1 expression and modulation in epithelial cells from nasal polyps. *Laryngoscope*. 2002;112(11):2067-2075. doi:10.1097/00005537-200211000-00030

32. Cho SH, Kim DW, Lee SH, et al. Age-related increased prevalence of asthma and nasal polyps in chronic rhinosinusitis and its association with altered IL-6 trans-signaling. *Am J Respir Cell Mol Biol*. 2015;53(5):601-606. doi:10.1165/rcmb.2015-0207RC

33. Klingberg TD, Pedersen MH, Cencic A, Budde BB. Application of measurements of transepithelial electrical resistance of intestinal epithelial cell monolayers to evaluate probiotic activity. *Appl Environ Microbiol*. 2005;71(11):7528-7530. doi:10.1128/AEM.71.11.7528-7530.2005

34. Carsuzaa F, Bequignon E, Bartier S, et al. Oncostatin M contributes to airway epithelial cell dysfunction in chronic rhinosinusitis with nasal polyps. *Int J Mol Sci*. 2023;24(7):6094. doi:10.3390/ijms24076094

35. Grosse-Onnebrink J, Werner C, Loges NT, et al. Effect of TH2 cytokines and interferon gamma on beat frequency of human respiratory cilia. *Pediatr Res*. 2016;79(5):731-735. doi:10.1038/pr. 2016.8

36. Bottier M, Blanchon S, Pelle G, et al. A new index for characterizing micro-bead motion in a flow induced by ciliary beating: part I, experimental analysis. *PLoS Comput Biol*. 2017;13(7):e1005605. doi:10.1371/journal.pcbi.1005605

37. Bottier M, Peña Fernández M, Pelle G, et al. A new index for characterizing micro-bead motion in a flow induced by ciliary beating: part II, modeling. *PLoS Comput Biol*. 2017;13(7):e1005552. doi:10.1371/journal.pcbi.1005552

38. Lechapt-Zalcman E, Prulière-Escabasse V, Advenier D, et al. Transforming growth factor-beta1 increases airway wound repair via MMP-2 upregulation: a new pathway for epithelial wound repair? *Am J Physiol Lung Cell Mol Physiol*. 2006;290(6):L1277-L1282. doi:10.1152/ajplung.00149. 2005

39. Boniface K, Diveu C, Morel F, et al. Oncostatin M secreted by skin infiltrating T lymphocytes is a potent keratinocyte activator involved in skin inflammation. *J Immunol*. 2007;178(7):4615-4622. doi:10.4049/jimmunol.178.7.4615

40. Junttila IS. Tuning the cytokine responses: an update on interleukin (IL)-4 and IL-13 receptor complexes. *Front Immunol*. 2018;9:888. doi:10.3389/fimmu.2018.00888

41. Zhang Y, Derycke L, Holtappels G, et al. Th2 cytokines orchestrate the secretion of MUC5AC and MUC5B in IL-5-positive chronic rhinosinusitis with nasal polyps. *Allergy*. 2019;74(1):131-140. doi:10.1111/all.13489

42. Van Zele T, Claeys S, Gevaert P, et al. Differentiation of chronic sinus diseases by measurement of inflammatory mediators. *Allergy*. 2006;61(11):1280-1289. doi:10.1111/j.1398-9995.2006.01225.x

43. Kariyawasam HH. Chronic rhinosinusitis with nasal polyps: insights into mechanisms of disease from emerging biological therapies. *Expert Rev Clin Immunol*. 2019;15(1):59-71. doi:10.1080/1744666X.2019.1541738

44. Chiba H, Osanai M, Murata M, Kojima T, Sawada N. Transmembrane proteins of tight junctions. *Biochim Biophys Acta*. 2008;1778(3):588-600. doi:10.1016/j.bbamem.2007.08.017

45. Wynne M, Atkinson C, Schlosser RJ, Mulligan JK. Contribution of epithelial cell dysfunction to the pathogenesis of chronic rhinosinusitis with nasal polyps. *Am J Rhinol Allergy*. 2019;33(6):782-790. doi:10.1177/1945892419868588

46. Babbin BA, Parkos CA, Mandell KJ, et al. Annexin 2 regulates intestinal epithelial cell spreading and wound closure through Rho-related signaling. *Am J Pathol*. 2007;170(3):951-966. doi:10.2353/ajpath.2007.060647

47. Kirshner J, Schumann D, Shively JE. CEACAM1, a cell-cell adhesion molecule, directly associates with annexin II in a three-dimensional model of mammary morphogenesis. *J Biol Chem*. 2003;278(50):50338-50345. doi:10.1074/jbc.M309115200

48. Hopkins AM, Bruewer M, Brown GT, et al. Epithelial cell spreading induced by hepatocyte growth factor influences paxillin protein synthesis and posttranslational modification. *Am J Physiol Gastrointest Liver Physiol*. 2004;287(4):G886-G898. doi:10.1152/ajpgi.00065.2004

49. Tos M, Mogensen C. Pathogenesis of nasal polyps. *Rhinology*. 1977;15(2):87-95.

50. Shi J, Fan Y, Xu R, et al. Characterizing T-cell phenotypes in nasal polyposis in Chinese patients. *J Investig Allergol Clin Immunol*. 2009;19(4):276-282.

51. Peters AT, Kato A, Zhang N, et al. Evidence for altered activity of the IL-6 pathway in chronic rhinosinusitis with nasal polyps. *J Allergy Clin Immunol*. 2010;125(2):397-403.e10. doi:10.1016/j.jaci. 2009.10.072

52. Derycke L, Eyerich S, Van Crombruggen K, et al. Mixed T helper cell signatures in chronic rhinosinusitis with and without polyps. *PLoS One*. 2014;9(6):e97581. doi:10.1371/journal.pone. 0097581

53. Chen X, Chang L, Li X, et al. Tc17/IL-17A up-regulated the expression of MMP-9 via NF-κB pathway in nasal epithelial cells of patients with chronic rhinosinusitis. *Front Immunol*. 2018;9:2121. doi:10.3389/fimmu.2018.02121

54. Carsuzaa F, Béquignon É, Bainaud M, et al. Oncostatin M counteracts the fibrotic effects of TGF-β1 and IL-4 on nasal-polyp-derived fibroblasts: a control of fibrosis in chronic rhinosinusitis with nasal polyps? *Int J Mol Sci*. 2022;23(11):6308. doi:10.3390/ijms23116308

55. Yan B, Lou H, Wang Y, et al. Epithelium-derived cystatin SN enhances eosinophil activation and infiltration through IL-5 in patients with chronic rhinosinusitis with nasal polyps. *J Allergy Clin Immunol*. 2019;144(2):455-469. doi:10.1016/j.jaci.2019.03.026
27. Steelant B, Seys SF, Boeckxstaens G, Akdis CA, Ceuppens JL, Hellings PW. Restoring airway epithelial barrier dysfunction: a new therapeutic challenge in allergic airway disease. *Rhinology*. 2016;54(3):195-205. doi:10.4193/Rhino15.376

28. Georas SN, Rezaee F. Epithelial barrier function: at the front line of asthma immunology and allergic airway inflammation. *J Allergy Clin Immunol*. 2014;134(3):509-520. doi:10.1016/j.jaci. 2014.05.049

29. Coste A, Rateau JG, Bernaudin JF, Peynègre R, Escudier E. Nasal polyposis pathogenesis: a flow cytometric and immunohistochemical study of epithelial cell proliferation. *Acta Otolaryngol (Stockh)*. 1996;116(5):755-761. doi:10.3109/00016489609137920

30. Coste A, Rateau JG, Roudot-Thoraval F, et al. Increased epithelial cell proliferation in nasal polyps. *Arch Otolaryngol Head Neck Surg*. 1996;122(4):432-436. doi:10.1001/archotol.1996. 01890160072013

31. Papon JF, Coste A, Gendron MC, et al. HLA-DR and ICAM-1 expression and modulation in epithelial cells from nasal polyps. *Laryngoscope*. 2002;112(11):2067-2075. doi:10.1097/00005537-200211000-00030

32. Cho SH, Kim DW, Lee SH, et al. Age-related increased prevalence of asthma and nasal polyps in chronic rhinosinusitis and its association with altered IL-6 trans-signaling. *Am J Respir Cell Mol Biol*. 2015;53(5):601-606. doi:10.1165/rcmb.2015-0207RC

33. Klingberg TD, Pedersen MH, Cencic A, Budde BB. Application of measurements of transepithelial electrical resistance of intestinal epithelial cell monolayers to evaluate probiotic activity. *Appl Environ Microbiol*. 2005;71(11):7528-7530. doi:10.1128/AEM.71.11.7528-7530.2005

34. Carsuzaa F, Bequignon E, Bartier S, et al. Oncostatin M contributes to airway epithelial cell dysfunction in chronic rhinosinusitis with nasal polyps. *Int J Mol Sci*. 2023;24(7):6094. doi:10.3390/ijms24076094

35. Grosse-Onnebrink J, Werner C, Loges NT, et al. Effect of TH2 cytokines and interferon gamma on beat frequency of human respiratory cilia. *Pediatr Res*. 2016;79(5):731-735. doi:10.1038/pr. 2016.8

36. Bottier M, Blanchon S, Pelle G, et al. A new index for characterizing micro-bead motion in a flow induced by ciliary beating: part I, experimental analysis. *PLoS Comput Biol*. 2017;13(7):e1005605. doi:10.1371/journal.pcbi.1005605

37. Bottier M, Peña Fernández M, Pelle G, et al. A new index for characterizing micro-bead motion in a flow induced by ciliary beating: part II, modeling. *PLoS Comput Biol*. 2017;13(7):e1005552. doi:10.1371/journal.pcbi.1005552

38. Lechapt-Zalcman E, Prulière-Escabasse V, Advenier D, et al. Transforming growth factor-beta1 increases airway wound repair via MMP-2 upregulation: a new pathway for epithelial wound repair? *Am J Physiol Lung Cell Mol Physiol*. 2006;290(6):L1277-L1282. doi:10.1152/ajplung.00149. 2005

39. Boniface K, Diveu C, Morel F, et al. Oncostatin M secreted by skin infiltrating T lymphocytes is a potent keratinocyte activator involved in skin inflammation. *J Immunol*. 2007;178(7):4615-4622. doi:10.4049/jimmunol.178.7.4615

40. Junttila IS. Tuning the cytokine responses: an update on interleukin (IL)-4 and IL-13 receptor complexes. *Front Immunol*. 2018;9:888. doi:10.3389/fimmu.2018.00888

41. Zhang Y, Derycke L, Holtappels G, et al. Th2 cytokines orchestrate the secretion of MUC5AC and MUC5B in IL-5-positive chronic rhinosinusitis with nasal polyps. *Allergy*. 2019;74(1):131-140. doi:10.1111/all.13489

42. Van Zele T, Claeys S, Gevaert P, et al. Differentiation of chronic sinus diseases by measurement of inflammatory mediators. *Allergy*. 2006;61(11):1280-1289. doi:10.1111/j.1398-9995.2006.01225.x

43. Kariyawasam HH. Chronic rhinosinusitis with nasal polyps: insights into mechanisms of disease from emerging biological therapies. *Expert Rev Clin Immunol*. 2019;15(1):59-71. doi:10.1080/1744666X.2019.1541738

44. Chiba H, Osanai M, Murata M, Kojima T, Sawada N. Transmembrane proteins of tight junctions. *Biochim Biophys Acta*. 2008;1778(3):588-600. doi:10.1016/j.bbamem.2007.08.017

45. Wynne M, Atkinson C, Schlosser RJ, Mulligan JK. Contribution of epithelial cell dysfunction to the pathogenesis of chronic rhinosinusitis with nasal polyps. *Am J Rhinol Allergy*. 2019;33(6):782-790. doi:10.1177/1945892419868588

46. Babbin BA, Parkos CA, Mandell KJ, et al. Annexin 2 regulates intestinal epithelial cell spreading and wound closure through Rho-related signaling. *Am J Pathol*. 2007;170(3):951-966. doi:10.2353/ajpath.2007.060647

47. Kirshner J, Schumann D, Shively JE. CEACAM1, a cell-cell adhesion molecule, directly associates with annexin II in a three-dimensional model of mammary morphogenesis. *J Biol Chem*. 2003;278(50):50338-50345. doi:10.1074/jbc.M309115200

48. Hopkins AM, Bruewer M, Brown GT, et al. Epithelial cell spreading induced by hepatocyte growth factor influences paxillin protein synthesis and posttranslational modification. *Am J Physiol Gastrointest Liver Physiol*. 2004;287(4):G886-G898. doi:10.1152/ajpgi.00065.2004

49. Tos M, Mogensen C. Pathogenesis of nasal polyps. *Rhinology*. 1977;15(2):87-95.

50. Shi J, Fan Y, Xu R, et al. Characterizing T-cell phenotypes in nasal polyposis in Chinese patients. *J Investig Allergol Clin Immunol*. 2009;19(4):276-282.

51. Peters AT, Kato A, Zhang N, et al. Evidence for altered activity of the IL-6 pathway in chronic rhinosinusitis with nasal polyps. *J Allergy Clin Immunol*. 2010;125(2):397-403.e10. doi:10.1016/j.jaci. 2009.10.072

52. Derycke L, Eyerich S, Van Crombruggen K, et al. Mixed T helper cell signatures in chronic rhinosinusitis with and without polyps. *PLoS One*. 2014;9(6):e97581. doi:10.1371/journal.pone. 0097581

53. Chen X, Chang L, Li X, et al. Tc17/IL-17A up-regulated the expression of MMP-9 via NF-κB pathway in nasal epithelial cells of patients with chronic rhinosinusitis. *Front Immunol*. 2018;9:2121. doi:10.3389/fimmu.2018.02121

54. Carsuzaa F, Béquignon É, Bainaud M, et al. Oncostatin M counteracts the fibrotic effects of TGF-β1 and IL-4 on nasal-polyp-derived fibroblasts: a control of fibrosis in chronic rhinosinusitis with nasal polyps? *Int J Mol Sci*. 2022;23(11):6308. doi:10.3390/ijms23116308

55. Yan B, Lou H, Wang Y, et al. Epithelium-derived cystatin SN enhances eosinophil activation and infiltration through IL-5 in patients with chronic rhinosinusitis with nasal polyps. *J Allergy Clin Immunol*. 2019;144(2):455-469. doi:10.1016/j.jaci.2019.03.026
**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:** Fieux M, Carsuzaa F, Bellanger Y, et al. Dupilumab prevents nasal epithelial function alteration by IL-4 in vitro: Evidence for its efficacy. *Int Forum Allergy Rhinol.* 2024;1-13. https://doi.org/10.1002/alg.23343
**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:** Fieux M, Carsuzaa F, Bellanger Y, et al. Dupilumab prevents nasal epithelial function alteration by IL-4 in vitro: Evidence for its efficacy. *Int Forum Allergy Rhinol.* 2024;1-13. https://doi.org/10.1002/alg.23343