Effects of Dupilumab on Itch-Related Events in Atopic Dermatitis: Implications for Assessing Treatment Efficacy in Clinical Practice

Dupilumab attenuates itch and skin inflammation in patients with atopic dermatitis (AD). However, itch-related events that are improved by dupilumab remain unclear. Therefore, the present study investigated changes in clinical scores, serum biomarkers, and the number of intraepidermal nerve fibers (IENFs) using skin biopsies and blood samples from 12 patients with moderate to severe AD before and after treatment with dupilumab. Clinical manifestations were assessed using eczema area and severity index (EASI) and visual analogue scale (VAS) scores at baseline and after 8 and 16 weeks of treatment. Serum levels of total immunoglobulin E (IgE), thymus and activation-regulated chemokine (TARC), interleukin (IL)-4, IL-13, IL-22, and IL-31 were examined by electrochemiluminescence, chemiluminescent enzyme immunoassays, ProQuantum immunoassays, and enzyme-linked immunosorbent assays (ELISA) at baseline and after 8 and 16 weeks of treatment. In skin biopsies from AD patients at baseline and after 16 weeks of treatment, IENFs were examined immunohistochemically with the anti-protein gene product (PGP) 9.5 antibody. The dupilumab treatment significantly improved EASI and VAS scores and decreased serum levels of TARC, IgE, and IL-22, whereas those of IL-13 and IL-31, and the number of IENFs remained unchanged and those of IL-4 increased. VAS scores were positively correlated with serum TARC, IL-22, and IgE levels and the degree of epidermal thickening. Serum IL-31 levels were positively correlated with the number of IENFs. These results suggest that serum TARC, IL-22, and IgE levels and epidermal thickness are itch-related events associated with dupilumab treatment and that serum IL-31 levels may reflect the degree of IENF density in AD patients. Therefore, dynamic changes may be used to assess the efficacy of dupilumab treatment to treat itching and inflammation in patients with AD.


Introduction
Atopic dermatitis (AD) is a common allergic inflammatory skin disease that is characterized by chronic and persisting itch and eczematous lesions and is one of the allergic diseases that include asthma and allergic rhinitis [1]. Two thirds of patients have moderate to severe symptoms and require systemic treatment [2]. The symptom burden has been suggested to include the daily effects associated with pruritus, such as sleep disturbance and poor performance at work [3]. AD markedly affects the quality of life of patients and their families [4,5].
Although many patients with AD respond to topical treatments, such as corticosteroids and calcineurin inhibitors, those with moderate to severe AD require systemic (g) Number of eosinophils in peripheral blood. Data are shown as the mean ± standard error of the mean (n = 12 per group). Statistical analyses were performed with Tukey's multiple comparison test. ** p < 0.01, *** p < 0.001, **** p < 0.0001. EASI, eczema area and severity index; VAS, visual analogue scale; TARC, thymus and activation-regulated chemokine; IgE, immunoglobulin E; ns, not significant.

The Severity of AD and Clinical Score of Lesional Skin
The severity of AD was assessed using EASI scores for dermatitis and VAS scores for itch at baseline and after 8 and 16 weeks of treatment (Figure 1a), according to previously reported methods [22,23].

Measurement of Serum Biomarkers
Blood samples were collected at baseline and after 8 and 16 weeks of treatment (Figure 1a). Serum levels of total IgE were measured using electrochemiluminescence. Serum TARC levels were assessed using a chemiluminescent enzyme immunoassay. The number of eosinophils was counted by an automatic analyzer method. Serum IL-4 and IL-13 levels were measured using a cytokine-specific ProQuantum Immunoassay kit (Invitrogen, Waltham, MA, USA), IL-22 by a Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minnesota, MN, USA), and IL-31 by the DuoSet ELISA kit (R&D Systems) according to the manufacturers' protocols.

Skin Biopsies
Punch biopsies of 3 mm in diameter were taken from lesional skin at baseline and after 16 weeks of treatment (Figure 1a). Post-treatment biopsy specimens were taken at the same location approximately 1 cm from prior biopsy scars. (g) Number of eosinophils in peripheral blood. Data are shown as the mean ± standard error of the mean (n = 12 per group). Statistical analyses were performed with Tukey's multiple comparison test. ** p < 0.01, *** p < 0.001, **** p < 0.0001. EASI, eczema area and severity index; VAS, visual analogue scale; TARC, thymus and activation-regulated chemokine; IgE, immunoglobulin E; ns, not significant.

The Severity of AD and Clinical Score of Lesional Skin
The severity of AD was assessed using EASI scores for dermatitis and VAS scores for itch at baseline and after 8 and 16 weeks of treatment (Figure 1a), according to previously reported methods [22,23].

Measurement of Serum Biomarkers
Blood samples were collected at baseline and after 8 and 16 weeks of treatment ( Figure 1a). Serum levels of total IgE were measured using electrochemiluminescence. Serum TARC levels were assessed using a chemiluminescent enzyme immunoassay. The number of eosinophils was counted by an automatic analyzer method. Serum IL-4 and IL-13 levels were measured using a cytokine-specific ProQuantum Immunoassay kit (Invitrogen, Waltham, MA, USA), IL-22 by a Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minnesota, MN, USA), and IL-31 by the DuoSet ELISA kit (R&D Systems) according to the manufacturers' protocols.

Skin Biopsies
Punch biopsies of 3 mm in diameter were taken from lesional skin at baseline and after 16 weeks of treatment ( Figure 1a). Post-treatment biopsy specimens were taken at the same location approximately 1 cm from prior biopsy scars.

Hematoxylin-Eosin (H&E) Staining
Skin biopsy samples were fixed with 4% paraformaldehyde at 4 • C for 4 h and immersed in 20% sucrose at 4 • C overnight. Samples were then embedded in optimal cutting temperature compound (Sakura Finetechnical Co., Tokyo, Japan) and frozen in liquid nitrogen. Five-micrometer-thick cryosections were cut using a CM1850 cryostat (Leica Microsystems, Wetzlar, Germany) and mounted on silane-coated glass slides. Sections were Cells 2023, 12, 239 4 of 11 stained with hematoxylin for 5 min followed by eosin for 1 min (all at room temperature). Sections were subsequently dehydrated with an ascending series (80, 95, and 100%) of ethanol at room temperature. Stained sections were observed under a BZ-X800 microscope (Keyence, Osaka, JAPAN). We measured epidermal thickness, avoiding the rete ridges, using a BZ-X800 analyzer (Keyence).

Semi-Quantification of IENFs
We counted the number of IENFs at the border between the dermis and epidermis. To semi-quantify the number of IENFs, 6 to 9 specimens per skin biopsy were stained with the anti-PGP9.5 antibody. Under a confocal microscope, the epidermal area at which PGP9.5immunoreactive fibers were the most abundant was selected in each specimen, and 0.9-µmthick optical sections were scanned through the z-plane of stained specimens (thickness of 25 µm). A three-dimensional reconstruction of images was performed with Leica Confocal software (Leica Microsystems). To measure the numbers of PGP9.5-immunoreactive fibers, 6 to 9 confocal images were analyzed in each biopsy specimen. The number of IENFs per 1.6 × 10 5 µm 2 in images was hand-counted by two researchers (R.K. and S.T.) in a not blinded manner. All values are presented as the means of three experiments.

Statistical Analysis
Data are shown as the mean ± standard error of the mean. Statistical analyses were performed with the unpaired t-test or a two-way ANOVA with Tukey's multiple comparison test or Šidák's multiple comparison test. Statistical analyses were conducted using Prism 9 software (Graphpad Software Inc., La Jolla, CA, USA). p < 0.05 was considered to be significant.

Characteristics of Participants
The characteristics of AD patients are shown in Table 1. The mean age of patients was 44 years and there were 8 males and 4 females. Mean EASI and VAS scores were 43.6 ± 3.7 and 72.5 ± 3.9, respectively.

Analyses of Clinical Scores in AD Patients before and after the Dupilumab Treatment
Clinical manifestations before and after 8 and 16 weeks of treatment are shown in Figure 1b. Scratch marks and lichenification on the back improved 8 and 16 weeks after treatment. EASI scores significantly decreased from baseline (43.6 ± 3.7) to week 16 (9.6 ± 0.9) (Figure 1c). EASI-75 was achieved by 75% of patients (8 out of 12). VAS scores also significantly decreased from baseline (72.5 ± 3.9) to week 16 (19.2 ± 5.5) (Figure 1d

Alterations in Serum Levels of Type 2 Cytokines in AD Patients before and after the Dupilumab Treatment
Serum IL-4 levels were higher after 8 and 16 weeks of treatment than at baseline (Figure 2a). Serum IL-13 levels were also higher after 8 weeks of treatment than at baseline, with no significant change after 16 weeks (Figure 2b). Serum IL-31 levels after 8 or 16 weeks of treatment did not significantly differ from those at baseline (Figure 2c).  Note: Data are shown as the mean ± standard error of the mean (n = 12 per group). Abbreviations: AD, atopic dermatitis; EASI, eczema area and severity index; VAS, visual analogue scale.

Analyses of Clinical Scores in AD Patients before and after the Dupilumab Treatment
Clinical manifestations before and after 8 and 16 weeks of treatment are shown in Figure 1b. Scratch marks and lichenification on the back improved 8 and 16 weeks after treatment. EASI scores significantly decreased from baseline (43.6 ± 3.7) to week 16 (9.6 ± 0.9) (Figure 1c). EASI-75 was achieved by 75% of patients (8 out of 12). VAS scores also significantly decreased from baseline (72.5 ± 3.9) to week 16 (19.

Alterations in Serum Levels of Type 2 Cytokines in AD Patients before and after the Dupilumab Treatment
Serum IL-4 levels were higher after 8 and 16 weeks of treatment than at baseline (Figure 2a). Serum IL-13 levels were also higher after 8 weeks of treatment than at baseline, with no significant change after 16 weeks (Figure 2b). Serum IL-31 levels after 8 or 16 weeks of treatment did not significantly differ from those at baseline (Figure 2c). Data are shown as the mean ± standard error of the mean (n = 12 per group). Statistical analyses were performed with Tukey's multiple comparison test. ** p < 0.01, *** p < 0.001, **** p < 0.0001. IL, interleukin; ns, not significant.

Analyses of Epidermal Thickness and Serum IL-22 Levels in AD Patients before and after the Dupilumab Treatment
H&E staining showed significant improvements in epidermal thickening after 16 weeks of treatment (Figure 3a

Evaluation of Factors for Treatment Effectiveness
VAS scores in AD patients were positively correlated with serum TARC, IL-22, and IgE levels and the degree of epidermal thickening, and EASI scores were positively correlated with serum TARC and IL-22 levels and the degree of epidermal thickening. These findings suggest that the dupilumab treatment correlated with a decrease in these parameters with improvements in VAS and EASI scores (Figures 4 and S1).

Evaluation of Factors for Treatment Effectiveness
VAS scores in AD patients were positively correlated with serum TARC, IL-22, and IgE levels and the degree of epidermal thickening, and EASI scores were positively correlated with serum TARC and IL-22 levels and the degree of epidermal thickening. These findings suggest that the dupilumab treatment correlated with a decrease in these parameters with improvements in VAS and EASI scores (Figures 4 and S1).

Evaluation of Factors for Treatment Effectiveness
VAS scores in AD patients were positively correlated with serum TARC, IL-22, and IgE levels and the degree of epidermal thickening, and EASI scores were positively correlated with serum TARC and IL-22 levels and the degree of epidermal thickening. These findings suggest that the dupilumab treatment correlated with a decrease in these parameters with improvements in VAS and EASI scores (Figures 4 and S1).

Distribution of IENFs in AD Patients before and after the Dupilumab Treatment
The distribution of IENFs in AD patients before and after the dupilumab treatment was examined by immunohistochemistry using an antibody to PGP9.5. IENFs decreased in two cases ( Figure 5a) and remained unchanged in eight cases after the dupilumab treatment (Figure 5b). In two cases, IENFs increased after the treatment (Figure 5c). In the semi-quantitative analysis of all participants, the number of IENFs remained unchanged before and after treatment (Figure 5d). Correlation analyses showed that serum IL-31 levels positively correlated with the number of IENFs (Figure 5e), whereas those of IL-4, IL-13, and IL-22 did not (Figure 5f-h).

Distribution of IENFs in AD Patients before and after the Dupilumab Treatment
The distribution of IENFs in AD patients before and after the dupilumab treatment was examined by immunohistochemistry using an antibody to PGP9.5. IENFs decreased in two cases ( Figure 5a) and remained unchanged in eight cases after the dupilumab treatment (Figure 5b). In two cases, IENFs increased after the treatment (Figure 5c). In the semiquantitative analysis of all participants, the number of IENFs remained unchanged before and after treatment (Figure 5d). Correlation analyses showed that serum IL-31 levels positively correlated with the number of IENFs (Figure 5e), whereas those of IL-4, IL-13, and IL-22 did not (Figure 5f-h).

Discussion
The present study showed that EASI scores, VAS scores, serum levels of TARC, IgE, and IL-22, and epidermal thickness decreased after the dupilumab treatment, whereas the number of eosinophils in peripheral blood, serum levels of IL-13 and IL-31, and the number of IENFs remained unchanged and serum levels of IL-4 increased ( Table 2). The primary outcome, EASI-75, which is used in clinical phase 3 AD trials, was achieved by 44-69% of patients after 16 weeks of treatment [10,11]. The present results revealed that 75% of patients (8 out of 12) achieved EASI-75, demonstrating the clinical effectiveness of dupilumab, which was consistent with the findings of clinical phase 3 AD trials. We also showed that the dupilumab treatment reduced serum levels of TARC and IgE, which are objective biomarkers for disease severity in AD [12,13].

Discussion
The present study showed that EASI scores, VAS scores, serum levels of TARC, IgE, and IL-22, and epidermal thickness decreased after the dupilumab treatment, whereas the number of eosinophils in peripheral blood, serum levels of IL-13 and IL-31, and the number of IENFs remained unchanged and serum levels of IL-4 increased ( Table 2). The primary outcome, EASI-75, which is used in clinical phase 3 AD trials, was achieved by 44-69% of patients after 16 weeks of treatment [10,11]. The present results revealed that 75% of patients (8 out of 12) achieved EASI-75, demonstrating the clinical effectiveness of dupilumab, which was consistent with the findings of clinical phase 3 AD trials. We also showed that the dupilumab treatment reduced serum levels of TARC and IgE, which are objective biomarkers for disease severity in AD [12,13]. The present results revealed that total eosinophil counts showed decreased tendency. In contrast, the findings of clinical trials of AD patients showed elevated eosinophil levels in peripheral blood during a treatment with dupilumab [24]. Dupilumab has been suggested to inhibit the migration of eosinophils into tissues by suppressing the IL-4-and IL-13induced production of eotaxins without affecting production or migration from the bone marrow [25]. However, the mechanisms underlying dupilumab-induced hypereosinophilia remain unknown. Moreover, a previous study showed that the number of eosinophils was significantly decreased in AD patients after 32 weeks of treatment with dupilumab [15]. Therefore, the attenuation of eosinophilia with dupilumab may require a longer treatment period than 16 weeks.
We also herein showed that serum levels of TARC, IL-22, and IgE and the degree of epidermal thickening positively correlated with the clinical scores VAS (degree of itching), and that serum levels of TARC and IL-22 and the degree of epidermal thickening were positively correlated with clinical scores of EASI (degree of dermatitis). Given that serum TARC and IL-22 levels correlated positively with VAS more strongly than with serum IgE level, serum TARC and IL-22 levels may be excellent parameters for assessing the efficacy of treating AD with dupilumab.
In the present study, the ProQuantum Immunoassay, a highly sensitive assay, revealed that serum IL-4 levels increased after 8 and 16 weeks of treatment and IL-13 levels after 8 weeks of treatment. A previous study reported increases in serum IL-4 and IL-13 levels in AD patients treated with dupilumab [16]. Furthermore, the blockade of IL-4 receptor α was recently shown to not affect the production of cytokines, such as thymic stromal lymphopoietin and IL-33, which stimulate group 2 innate lymphoid cells and activate Th2 cells [26,27]. This may at least partly explain why serum levels of IL-4 and IL-13 were not normalized by the dupilumab treatment. In a clinical trial on AD patients treated with dupilumab, symptoms worsened soon after the discontinuation of dupilumab [28]. Another possibility might be compensated for by inhibiting the IL-4/IL-13 signaling pathway [29]. Therefore, since dupilumab blocks IL-4 receptor α and inhibits IL-4 and IL-13 signaling pathways, serum levels of IL-4 and IL-13 have potential as parameters to assess the termination of dupilumab therapy; however, it is not considered possible to stop dupilumab at 16 weeks. Further studies are needed to investigate the dynamics of IL-4 and IL-13 in the serum of AD patients after 16 weeks of treatment. In the present study, we examined serum levels of IL-4 and IL-13; however, their expression in tissue may also change. Therefore, further clinical investigations are needed.
Th2 cells secrete IL-31 as an itch mediator and/or modulator, which acts on sensitized sensory nerves and induces intense itching [30,31]. IL-31 has been also shown to promote sensory nerve fiber outgrowth in vitro [32]. Serum levels of IL-31 positively correlated with the number of IENFs (Figure 5e). Therefore, serum levels of IL-31 may be used as a parameter to assess the status of intraepidermal nerve density Previous studies have reported that IL-4 receptor α/γc heterodimer is expressed on lymphocytes, and when IL-4 binds to it, they induce differentiation into Th2 cells and production of TARC [33,34]. TARC released from Th2 cells then binds to C-C chemokine receptor 4 (CCR4) expressed on Th22 cells and recruits Th22 cells to the lesional skin to produce IL-22, which binds to IL-22 receptors expressed on keratinocytes to induce acanthosis [35]. Thus, IL-22 is a cytokine that induces keratinocyte proliferation [36]. In the present study, we found that epidermal thickness and serum IL-22 levels decreased after the dupilumab treatment, and also that epidermal thickness positively correlated with IL-22 levels ( Figure 3). Dupilumab has been previously shown to reduce epidermal thickness [37]. Reductions in both the serum and transcription levels of IL-22 were recently reported in AD treated with dupilumab [16,37]. A previous study reported that Th22 cells producing IL-22 expressed CCR4, which is targeted by TARC [38]. Furthermore, serum levels of TARC correlated with those of IL-22 [39]. Based on these findings, a decrease in TARC levels may suppress the production of IL-22 by Th22 cells. Taken together, although the precise mechanisms by which the dupilumab treatment reduced serum IL-22 levels remain unclear, these findings suggest that IL-4 receptor α signaling at least partly affects IL-22 production.

Conclusions
In conclusion, the present results revealed that the dupilumab treatment improved EASI and VAS scores and decreased serum levels of TARC, IgE, and IL-22, whereas serum levels of IL-13 and IL-31 and the number of eosinophils in peripheral blood remained unchanged and serum levels of IL-4 increased. VAS scores were positively correlated with serum TARC, IL-22, and IgE levels and the degree of epidermal thickening, and EASI scores were positively correlated with serum TARC and IL-22 levels and the degree of epidermal thickening. Moreover, serum IL-31 levels positively correlated with the number of IENFs. These results suggest that serum TARC, IL-22, and IgE levels and epidermal thickness are itch-related events associated with dupilumab, and also that serum IL-31 levels may reflect the degree of IENF density in patients with AD. Therefore, dynamic changes may be used to assess the efficacy of dupilumab to treat AD.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethical Committee of Juntendo University Urayasu Hospital (approval number: U13-0003-U01).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.