Evaluation of innate lymphoid cells and their subsets in atopic dermatitis patients with and without dupilumab therapy

Dear Editor,

In atopic dermatitis (AD) patients, innate lymphoid cells (ILCs) are not examined as well as T cells. It is not yet known what is the count of innate lymphoid cells (ILCs) and their types (ILC −1, ILC-2, ILC-3, ILC-3 NCR+, ILC-3 NCR-) in AD patients with and without dupilumab therapy.

ILCs are a population of lymphoid cells that do not express T cell or B cell antigen-specific receptors. They are largely tissue-resident and enriched at mucosal sites to play a protective role against pathogens. ILCs mimic the functions of CD4 T helper (Th) subsets (1,2). ILCs have been categorized into three different groups, ILC-1, ILC-2, and ILC-3, based on their distinct pattern of cytokine production and transcription factors. Type 1 innate lymphoid cells (ILC-1) are defined by the expression of signature cytokine IFN-γ and the master transcription factor T-bet, involving in the type 1 immune response. Type 2 innate lymphoid cells (ILC-2) can induce a type 2 inflammatory response. ILC-2s share many functional similarities with Th2 lymphocytes because they produce type 2 cytokines interleukin (IL)- 4, IL-5, IL-9, and IL-13 as well as other effector molecules. Type 3 innate lymphoid cells (ILC-3) are defined by their ability to produce IL-17 and/or IL-22 and can be further subclassified as lymphoid tissue-inducer (LTi) cells, natural cytotoxicity triggering receptor-positive (NCR)⁺ ILC3 cells, and NCR⁻ILC3 cells (3). ILC subsets mainly reside in tissues and represent an early source of cytokines in response to infections or tissue damage. A tumor-promoting role has recently been proposed for ILC3-derived IL-22 in colorectal cancer (4). In the skin, ILC are well placed to sense keratinocyte-derived danger signals in an antigen-independent manner. Recent findings link ILC-2 to atopic dermatitis and ILC-3 to psoriasis (1,2).

Dupilumab, a monoclonal antibody is designed to block IL-4 and IL-13 receptors. By blocking these receptors, dupilumab prevents the action of IL-4 and IL-13 and alleviates the symptoms of atopic dermatitis (3). In addition to the IL-4 receptor, IL-13 binds to a lesser-known receptor, the IL-13 α2 subunit receptor (IL-13Rα2), of which the function remains unclear. It has been postulated that this receptor acts as a ‘decoy’ by binding to the cytokine without downstream effects, reducing IL-13 levels in the serum and further inhibiting the IL-4 pathway (4).

The aim of the study is to evaluate the count of ILCs and their types (ILC −1, ILC-2, ILC-3, ILC-3 NCR+, ILC-3 NCR-) in AD patients with and without dupilumab therapy and evaluate the relation between the severity of AD and the count of ILCs and their types (ILC −1, ILC-2, ILC-3, ILC-3 NCR+, ILC-3 NCR-).

In our study, ILC cells were examined in 77 AD patients (37 men and 40 women) with a mean age of 46.6 years. This group consisted of 20 patients treated with dupilumab (6 women and 14 men) at a mean age of 45.3 years and 57 patients without dupilumab treatment (34 women and 23 men) at a mean age of 46.1 years. In patients with dupilumab, the mild form of AD was recorded in 17 patiens and moderate form in three patients. In patients without dupilumab therapy, 12 patients suffer from mild form, 34 from moderate form and 11 from severe form. At the same time, 40 healthy subjects were examined with this study group, which consisted of 22 women and 18 men with a mean age of 42.3 years.

Complete dermatological examination was performed in all patients included in the study.

All these patients were examined in the Department of Dermatology, Faculty Hospital Hradec Králové, Charles University, Czech Republic. The diagnosis of AD was determined according to Hanifin-Rajka’s diagnostic criteria. Inclusion criteria: (1) age 14 years and over (2) AD as defined by the criteria of Hanifin and Rajka. Patients with moderate and severe form of AD without dupilumab and patients with dupilumab therapy lasting at least 18 months were included. Exclusion criteria: pregnancy, breastfeeding, systemic therapy (cyclosporin, systemic corticoids). The severity of AD was evaluated with EASI and SCORAD index.

Surface molecules expressed on immune cells were analyzed by flow cytometry using specially prepared tubes containing monoclonal antibodies (DURAClone IM Innate Lymphoid Cell Tube, Beckman Coulter). For each patient and control group, one indicated tube was analyzed. The gating strategy for ILC cells and their subsets is recorded in Table 1.

Table 1. Gating strategies for ILC cells and their subsets (ILC-1, ILC-2, ILC-3, ILC-3 NCR+, ILC-3 NCR.

Gating strategies for ILC cells	Negativity of the markers: CD1a, CD3, CD14, CD16, CD19, CD34, CD94, CD123, TCRα/β, TCRγ/δ, FαR1a (LIN)
	Character positivity: CD45, CD127
ILC-1 cells	CD1a, CD3, CD14, CD16, CD19, CD34, CD94, CD123, TCRα/β, TCRγ/δ, FαR1a (LIN) and CD117.
	Sign negativity: CD335 (NKp46)
ILC-2 cells	Character negativity: CD1a, CD3, CD14, CD16, CD19, CD34, CD94, CD123, TCRα/β, TCRγ/δ, FαR1a (LIN)
	Character positivity: CD294, CD161
ILC-3 cells	Character negativity: CD1a, CD3, CD14, CD16, CD19, CD34, CD94, CD123, TCRα/β, TCRγ/δ, FαR1a (LIN)
	Character positivity: CD117

The laboratory results (the count of lymphocytes, total ILCs, ILC −1, ILC-2, ILC-3, NCR + ILC3, NCR⁻ILC3) were compared in AD patients (treated or not treated with dupilumab) and in control group. We compared also the severity of AD with the count of ILC cells and their subsets in both groups of AD patients. For statistical analysis, we used nonparametric Kruskal–Wallis one-factor analysis of variance with post hoc (follow-up multiple comparison) and Dunn’s test with Bonferroni modification of significance level. We used statistical software: NCSS 2021 Statistical Software (2021). NCSS, LLC. Kaysville, Utah, USA, ncss.com/software/ncss.

The basic characteristics of patients treated and not treated with dupilumab are shown in Table 2. The representation of AD patients (treated or nontreated with dupilumab) did not differ in terms of age, gender and onset of AD. Likewise, the representation of the control group did not differ in terms of age and gender. AD severity did not differ between the two groups of patients with AD before starting dupilumab therapy. Patients treated with dupilumab had moderate and severe AD before starting biologic therapy; after starting dupilumab, skin findings improved significantly (Table 2). In addition to dupilumab, which is applied 300 mg s.c. every two weeks, they are treated with topical therapy to hydrate the skin. Patients without dupilumab therapy are treated with emollients, topical corticosteroids with antispetics for acute exacerbations.

Table 2. Characteristic of atopic dermatitis patients.

Patient characteristics	Dupilumab untreated patients	Dupilumab treated patients
Age	46.1 years (24.2- 52.3)	45.3 years (31.6-48.3)
Number of patients	57 (34 women and 23 men)	20 (14 men, 6 women)
SCORAD	31.5 (26.5-38.7)	35.1 (30.5-45.2)	Before dupilumab therapy
		9.5 (7.1-18.2)	Average value after 1.5 years of treatment with dupilumab
EASI	31.3 (26.8-38.5)	33.1 (30.1-44.2)	Before dupilumab therapy
		9.1 (8.2-17.2)	Average value after 1.5 years of treatment with dupilumab
POEM	13.6 (10-18)	15.3 (12–21)	Before dupilumab therapy
		4.1 (2–6)	Average value after 1.5 years of treatment with dupilumab
DLQI	12.6 (9 – 16)	15.5 (11-20)	Before dupilumab therapy
		3.4 (1–5)	Average value after 1.5 years of treatment with dupilumab

Control group - 40 healthy subjects (18 men, 22 women), age 42.3 years s.d. 9.2 years.

The count of total lymphocytes, ILC cells and their subsets (ILC −1, ILC-2, ILC-3, NCR + ILC3, NCR⁻ILC3 in AD patients (with and without dupilumab therapy) is recorded in Table 3. Our results show that:

Table 3. The count of total lymphocytes, ILC cells and their subsets (ILC-1, ILC-2, ILC-3, ILC-3 NCR+, ILC-3 NCR-) in AD patients and control group, statistical analysis.

	Median values (10^9/l)			Statistical analysis
	DUP-	DUP+	Control group	DUP+/DUP p - value	DUP-/control p - value	DUP+ /control p - value
Lymphocytes 10^9/l	1.67	1.75	1.79
Total ILC 10^9/l	0.0011	0.0015	0.0016		˂ 0.05
ILC-1 10^9/l	0.00035	0.00033	0.00037
ILC-2 10^9/l	0.00027	0.00042	0.00042		˂ 0.05
ILC-3 10^9/l	0.00045	0.00052	0.00072
ILC-3 NCR+ 10^9/l	0.00040	0.00048	0.00061
ILC-3 NCR- 10^9/l	0.000037	0.000054	0.000082		˂ 0.001

1. The count of total lymphocytes does not significantly differ in AD patients (with and without therapy) compared to control group.

2. The significant changes in ILCs were recorded only in AD patients without dupilumab therapy. We confirmed the significantly lower count of total ILC cells, ILC-2 and NCR⁻ILC3 in AD patients without dupilumab therapy compared to control group. 3) The count of ILC-1 and ILC-3 and NCR + ILC3 does not significantly differ in AD patients (with and without dupilumab therapy) from control group.

The relation between the count of total lymphocytes, ILC cells and their subsets (ILC −1, ILC-2, ILC-3, NCR + ILC3, NCR⁻ILC3 in AD patients without dupilumab therapy is recorded in Table 4 a). The relation between the count of total lymphocytes, ILC cells and their subsets (ILC −1, ILC-2, ILC-3, NCR + ILC3, NCR⁻ILC3 in all AD patients (with and without dupilumab therapy) is recorded in Table 4 b). We show no difference between ILCs and their subsets in mild, moderate and severe AD in patients without dupilumab therapy and as well in all included AD patients (with and without dupilumab therapy). The severity of atopic dermatitis is not significantly related to the count of ILC cells and their subsets.

Table 4a. AD severity and the count of ILC cells in AD patients without dupilumab therapy. The relation between AD severity and the count of ILCs and their subsets was not confirmed (≥ 0.05).

	Severity of AD, number of patients			Statistical analysis
	Mild form 12 patients	Moderate form 34 patients	Severe form 11 patients	Mild/moderate p - value	Mild/severe p - value	Moderate/severe p - value
Lymphocytes 10^9/l	1.62075	1.61205	2.2125	≥ 0.05	≥ 0.05	≥ 0.05
Total ILC 10^9/l	0.00128	0.00107	0.00111	≥ 0.05	≥ 0.05	≥ 0.05
ILC-1 10^9/l	0.00036	0.00036	0.00031	≥ 0.05	≥ 0.05	≥ 0.05
ILC-2 10^9/l	0.00026	0.00028	0.00014	≥ 0.05	≥ 0.05	≥ 0.05
ILC-3 10^9/l	0.00047	0.00047	0.00027	≥ 0.05	≥ 0.05	≥ 0.05
ILC-3 NCR+ 10^9/l	0.00041	0.00042	0.00024	≥ 0.05	≥ 0.05	≥ 0.05
ILC-3 NCR- 10^9/l	5.0116E-05	3.6642E-05	2.8644E-05	≥ 0.05	≥ 0.05	≥ 0.05

Table 4b. AD severity and the count of ILC cells in all AD patients (with and without dupilumab therapy). the relation between AD severity and the count of ILCs and their subsets was not confirmed (≥ 0.05).

	Severity of AD, number of patients			Statistical analysis
	Mild 29 patients	Moderate 37 patients	Severe 11 patients	Mild/moderate p - value	Mild/severe p - value	Moderate/severe p - value
Lymphocytes 10^9/l	1.638	1.6425	2.2125	≥ 0.05	≥ 0.05	≥ 0.05
Total ILC 10^9/l	0.00132	0.00111	0.00112	≥ 0.05	≥ 0.05	≥ 0.05
ILC-1 10^9/l	0.00035	0.00035	0.00031	≥ 0.05	≥ 0.05	≥ 0.05
ILC-2 10^9/l	0.00027	0.00028	0.00014	≥ 0.05	≥ 0.05	≥ 0.05
ILC-3 10^9/l	0.00049	0.00047	0.00027	≥ 0.05	≥ 0.05	≥ 0.05
ILC-3 NCR+ 10^9/l	0.00041	0.00042	0.00025	≥ 0.05	≥ 0.05	≥ 0.05
ILC-3 NCR- 10^9/l	5.0184E-05	3.7016E-05	2.8644E-05	≥ 0.05	≥ 0.05	≥ 0.05

According to our results, the level of ILC-2 cells in patients treated with dupilumab does not differ from the control group. Before starting dupilumab therapy, these patients suffered from severe form of AD. During dupilumab treatment skin findings improved significantly and they now have a mild form of AD. On the contrary, there is a significant decrease of ILC-2 cells in peripheral blood in patients without dupilumab treatment; these patients suffer from moderate and severe form. We hypothesize that in AD patients suffering from moderate and severe form ILC-2 cells migrate to the skin lesions at an increased rate and thus are reduced in the peripheral blood. Alkon et col. investigated phenotypic and molecular features of ILCs under physiologic, normal human skin and pathologic, lesional skin of patients with AD conditions. Skin punch biopsies and reduction sheets as well as blood specimens were obtained from either patients with AD or healthy individuals. They found that the vast majority of cutaneous ILCs belong to reside in the upper skin layers. Single-cell RNA sequencing of cutaneous ILC-enriched cell samples confirmed the predominance of biologically heterogeneous group 2 ILCs and, for the first time, demonstrated considerable ILC lineage infidelity in lesional AD skin, and to a much lesser extent, in normal human skin. Similar events were demonstrated in ILCs from skin explant cultures and in vitro expanded ILCs from the peripheral blood (6).

Another interesting result of our study is the finding of the significantly lower count of NCR⁻ILC3 in AD patients without dupilumab therapy. The NCR⁻ILC3 is a heterogeneous subgroup that also contains lymphoid tissue inducers cells and is one of the important sources of IL-17 (7–9). The properties of tissue-resident ILC3 have been reported for intestine, skin, and lung (8,9). Co-culture experiment of neutrophils with NCR^- ILC3s revealed that NCR^- ILC3s stimulate neutrophils by producing granulocyte–macrophage colony-stimulating factor (GM-CSF), (10). Another study found that ILC3 lacking the natural cytotoxicity-triggering receptor (NCR⁻ILC3) promoted the development of hepatocellular carcinoma in response to interleukin 23 (11). Furthermore, NCR^-ILC3 initiated IL-17 production upon IL-23 stimulation and directly inhibited CD8⁺ T cell immunity by promoting lymphocyte apoptosis and limiting their proliferation (11). The specific mechanisms leading to the reduction in NCR − ILC3 cells in peripheral blood in atopic dermatitis are not fully understood, but it’s thought to be related to dysregulation of the immune response in the skin. We can assume that, like ILC-2, the NCR⁻ILC3 cells migrate to the affected tissue and are therefore reduced in peripheral blood.

Results of our study support the concept that the skin immune system consists of a network of highly flexible cellular players, such as ILC cells, that are capable of adjusting their function to the needs and challenges of the environment (6).

In conclusion, although we showed that the number of ILC cells, ILC-2 and NCR⁻ILC3 cells was significantly lower in AD patients without dupilumab, we did not demonstrate a difference between the count of ILCs and their subsets in mild, moderate and severe AD.

AD patients treated with dupilumab with improving the severity of AD have normalized total ILC cells, ILC-2 and NCR⁻ILC3 cells in peripheral blood. We can assume that the significantly low level of these cells in the peripheral blood of patients without dupilumab therapy is due to the migration of these cells into the affected skin. Further studies are needed to confirm this finding.

Jarmila Čelakovská
Department of Dermatology and Venereology Faculty Hospital, and Medical Faculty of Charles University, Hradec Králové, Czech [email protected] ČermákováDepartment of Medical Biophysics, Medical Faculty of Charles University, Hradec Králové, Czech RepublicPetra Boudkova and Jan KrejsekDepartment of Clinical Immunology and Allergy, Faculty Hospital and Medical Faculty of Charles University, Hradec Králové, Czech RepublicReceived 20 December 2023; Accepted 21 December 2023

Author Contribution

Assoc. Prof. Jarmila Čelakovská, M.D., Ph.D – the main investigator

Department of Dermatology and Venereology Faculty Hospital and Medical Faculty of Charles University, Hradec Králové, Czech Republic

• selection of the patients, dermatological examination and recommendations for the laboratory examination

• processing of all results

• writing of the manuscript

RNDr. Eva Čermáková

Department of Medical Biophysics, Medical Faculty of Charles University, Hradec Králové, Czech Republic

• statistical analysis

Petra Boudková, MSc. Eng.

Department of Clinical Immunology and Allergy, Faculty Hospital and Medical Faculty of Charles University, Hradec Králové, 50002, Czech Republic

• laboratory examination, flow cytometry

Prof. RNDr. Ctirad Andrýs, CSc

Department of Clinical Immunology and Allergy, Faculty Hospital and Medical Faculty of Charles University, Hradec Králové, 50002, Czech Republic

• control of laboratory examination

Prof. RNDr. Jan Krejsek CSc

Department of Clinical Immunology and Allergy, Faculty Hospital and Medical Faculty of Charles University, Hradec Králové, 50002, Czech Republic

professional cooperation, supervision

This study is approved by Ethics committee

This study was approved by Ethics committee of the Faculty Hospital Hradec Králové, Charles University of Prague, Czech Republic. Reference number is: 2021 10 P 03. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board - Ethics committee of the Faculty Hospital Hradec Králové, Charles University of Prague, Czech Republic. Data of Approval 4 September, 2021.

Informed consent was obtained from all subjects involved in the study.

Medical writing/Editorial assistance

The article was written by the main author, no other services and Editorial assistence were needed.

Prior publication

This manuscript is not based on work that has been previously presented/ published.

Authorship

All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published.

Explanation: The average values (minimal, maximal values) of SCORAD, EASI, POEM, DLQI are recorded. SCORAD – Scoring of atopic dermatitis, EASI -Eczema Area and Severity Index, POEM - Patient Oriented Eczema Measure, DLQI - Dermatology Life Quality Index.

Explanation: The count of ILCs and their subsets in AD patients with and without dupilumab therapy compared to control group. The median value is recorded. DUP – patients without dupilumab treatment, DUP + patients with dupilumab treatment. ILC innate lymphoid cells, ILC −1, ILC-2, ILC-3, ILC-3 NCR+, ILC-3 NCR- subsets of ILCs. We show the significant difference in statistical analysis, p – value < 0.05. An empty field for the p- value means that no significant difference was found.

Disclosure statement

Jarmila Čelakovská, Eva Čermákova, Petra Boudková, Ctirad Andrýs, Jan Krejsek have nothing to disclose.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Additional information

Funding

Charles University, Medical Faculty Hradec Králové, Cooperation, INDI 207034 (the financial support was intended for research purposes to examine the immunological profile). Supported by MH CZ - DRO (UHHK, 00179906). The Service Fee was funded by the authors.