Dear Editor,
Subsets of white blood cells are considered as the central component in immune-mediated diseases, such as atopic dermatitis AD (Citation1). CD4+ and, to some extent, also CD8+ T cells can differentiate into several effector subsets according to their pattern of cytokine expression, including the T helper 1 (Th1), Th2, and Th17 cells (Citation1). Eosinophil numbers in peripheral blood are elevated in most AD patients and appear to correlate with disease activity (Citation2). Basophils exacerbate Th2 cell differentiation, they infiltrate the skin to innate lymphoid cells type 2 (ILC2) expansion and are responsible for the rapid secretion of IL-4 and IL-13 (Citation3). The markers CD16 for eosinophils and CD203 for basophils have been used in recent years to assess their activation. Suppression of inflammatory changes in AD is demonstrated in the case of dupilumab therapy, which inhibits the production of key cytokines IL-4 and IL-13 (Citation4).
In this letter, we would like to build on our previous results and complete the immunological profile of patients with atopic dermatitis (Citation5,Citation6). In our previous studies, we evaluated in the same group of atopic dermatitis patients the count of B lymphocytes (CD5, CD22, CD73 B lymphocytes) and expression of activation markers CD200 and CD23 on B lymphocytes in the pollen season and outside the pollen season (Citation5,Citation6).
In this communication, we present the count of leukocytes, lymphocytes, neutrophils, monocytes, eosinophils and we report for the first time the count of CD16+ eosinophils, basophils, CD203+ basophils, T lymphocytes, natural killer cells (NK cells), CD4+ T lymphocytes, CD8+ T lymphocytes and T regulatory lymphocytes in patients suffering from moderate and severe form of AD (with and without dupilumab therapy) and in healthy subjects in pollen season (June–August) and out of pollen season (October–February).
During pollen season and out of pollen season, we examined two groups of AD patients; one group consisted of patients suffering from moderate and severe form of AD without dupilumab treatment (21 patients − 6 men, 15 women, age 36.2 years) and the other group consisted of patients treated with dupilumab at least 18 months (13 patients − 7 men, 6 women, age 43.4 years). All included patients underwent complete dermatological examination in the Department of Dermatology, Faculty Hospital Hradec Králové, Charles University, Czech Republic. Exclusion criteria were pregnancy, breastfeeding, systemic therapy (cyclosporin, systemic corticoids). Laboratory tests to determine the parameters of interest were performed in these patients during the pollen season (June-August) and outside the pollen season (October–February). The representation of AD patients was identical in both these periods. As a control group we included forty healthy individuals in pollen season and thirty healthy individuals out of pollen season, all blood donors with negative total IgE and with no signs of allergy. The representation of the control group matched with regard to gender and age to AD patients.
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 and they now have a mild form of AD. 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 immunomodulators and topical corticosteroids with antiseptics for acute exacerbations.
For the evaluation of pollen allergy, we analyzed the results of specific IgE to molecular components of pollen in all AD patients (with and without dupilumab therapy) included in the study. The specific IgE was examined with the use of ALEX2 Allergy Xplorer test. The determination of the number of leukocytes and lymphocytes within the leukocyte differential, was performed on Sysmex XN-10 blood cell analyzers using a combination of hydrodynamic focusing and flow fluorescence cytometry. T lymphocytes were evaluated with flow cytometry.
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. The Wilcoxon test for pairwise comparisons between AD patients and the Mann–Whitney and Kolmogorov–Smirnov test for controls were used. We used statistical software: NCSS 2021 Statistical Software.
The characteristic of AD patients (AD severity - EASI, SCORAD, the quality of life, the results of specific IgE to molecular components of pollen) is recorded in . The pollen allergy was confirmed in patients treated with dupilumab in 93.3% and in patients without dupilumab in 66.7% (Citation5,Citation6).
Table 1. Characteristic of patients.
The absolute and relative count of leukocytes, lymphocytes, neutrophils and monocytes in AD patients with and without dupilumab therapy in pollen season and out of pollen season is recorded in . The absolute and relative count of eosinophils, relative CD16+ eosinophils, the absolute and relative count of basophils and relative CD203+ basophils in AD patients with and without dupilumab therapy in pollen season and out of pollen season is recorded in . The absolute and relative count of T lymphocytes (CD4+, CD8+, regulatory T lymphocytes) and NK cells in AD patients with and without dupilumab therapy in pollen season and out of pollen season is recorded in .
Table 2. The absolute and relative count of leukocytes, lymphocytes, neutrophils and monocytes in AD patients with and without dupilumab therapy in pollen season and out of pollen season.
Table 3. The absolute and relative count of eosinophils, relative CD16+ eosinophils, the absolute and relative count of basophils and relative CD203+ basophils in AD patients with and without dupilumab therapy in pollen season and out of pollen season.
Table 4. The absolute and relative count of T lymphocytes (CD4+, CD8+, regulatory T lymphocytes) and NK cells in AD patients with and without dupilumab therapy in pollen season and out of pollen season.
The review of monitored parameters in AD patients with and without dupilumab therapy in pollen season and out of pollen season in comparison to control group is recorded in . The intercomparison of monitored parameters in AD patients with and without dupilumab therapy in pollen season compared to the count out of pollen season is recorded in .
Table 5. The review of monitored parameters in AD patients with and without dupilumab therapy in pollen season and out of pollen season in comparison to control group.
Table 6. The intercomparison of monitored parameters in AD patients with and without dupilumab therapy in pollen season compared to the count out of pollen season.
Our study shows interesting results: When we compared the observed parameters with respect to the control group in both periods (horizontal comparison in ), we found the following results:
In both seasons compared to control group, we confirmed the significantly higher count of neutrophils in AD patients without dupilumab therapy. In patients treated with dupilumab, we confirmed the lower count of CD8+ T lymphocytes, the higher count of monocytes, the lower count of relative basophils and the lower count of CD203+ basophils. In both groups of AD patients, we confirmed the higher count of eosinophils.
In pollen season compared to control group, we confirmed in both groups of AD patients the significantly higher count of absolute monocytes and the higher count of absolute basophils. In patients treated with dupilumab, we confirmed the higher count of CD16+ eosinophils and the higher count of relative T regulatory lymphocytes.
Out of pollen season compared to control group, we confirmed in AD patients with dupilumab the significantly higher count of neutrophils and relative CD4+ T lymphocytes. In AD patients without dupilumab, we confirmed the higher count of relative eosinophils.
The significant changes in immunological parameters in AD patients and in control group are evident when comparing them to pollen season and out of pollen season (vertical comparison in ).
1) In control group during pollen season, we recorded the lower count of monocytes, eosinophils, CD16+ eosinophils, and basophils compared to the period out of pollen season. On the other hand, the count of T lymphocytes is in control group in pollen season significantly higher in comparison to out of pollen season.
2) In patients with dupilumab therapy, we observe in pollen season the significant decrease of absolute and relative monocytes and relative eosinophils.
3) In AD patients without dupilumab therapy, we confirmed in pollen season the decrease of relative monocytes, absolute and relative eosinophils, the decrease of NK cells and the increase of T regulatory lymphocytes compared to out of season.
We demonstrated several statistically significant differences in our study in AD patients (treated and untreated with dupilumab) compared to controls in pollen and out of pollen season. As the most significant we consider the results in AD patients treated with dupilumab, such as the lower count of CD8+ T lymphocytes, higher count of absolute monocytes and eosinophils and the lower count of activated basophils (CD203+ basophils) in both seasons. In addition, the higher count of relative CD4+ T lymphocytes and the higher count of activated eosinophils (CD16+ eosinophils) in pollen season in patients with dupilumab therapy was recorded. It is well established that CD8+ T cells constitute an important branch of adaptive immunity contributing to clearance of intracellular pathogens and providing long-term protection. These functions are mostly fulfilled by the best characterized subpopulation of CD8+ T cells, the cytotoxic T lymphocytes (also called Tc1 cells), owing to their ability to kill infected cells and to secrete cytokines such as interferon-γ and tumor necrosis factor-α (Citation7). However, there is growing evidence for alternative CD8+ T cell fates influencing CD4+ T-cell-mediated responses in the context of allergy, autoimmunity and infections (Citation8).
The reduction of CD203 + basophil count may contribute to the therapeutic effects of dupilumab by reducing the inflammatory response and allergic reactions in patients with AD, because basophils are responsible for the rapid secretion of IL-4 and IL-13.
Another interesting finding is the higher count of activated eosinophils (CD16+ eosinophils) in pollen season in AD patients with dupilumab therapy. Blood eosinophils have mRNA for FcγRIIIB (CD16) but no or minimal spontaneous CD16 expression, and they express CD16 on their surface when stimulated in vitro with platelet-activating factor or IFN gamma. Transient expression of CD16 is also observed in vivo following aeroallergen challenge of asthmatic subjects (Citation9). An explanation for the higher count of activated eosinophils may be the high prevalence of pollen allergy in dupilumab patients (93.3%). In our previous study, we confirmed the higher association between the count of eosinophils (absolute and relative) and the expression of CD23 marker on B cells in patients with AD under dupilumab therapy. It suggests that IL-4 production by eosinophils may play a role in B lymphocyte activation (Citation10).
Surprisingly, we found that AD patients have an increase in eosinophils and monocytes in out of pollen season, which may be related to the fact that respiratory infections are more frequent in winter. Eosinophils cooperate significantly with B lymphocytes and participate in the immune response against respiratory viruses such as respiratory syncytial virus and influenza (Citation11). Another reason for the low levels of eosinophils and monocytes in peripheral blood of AD patients during the pollen season may be that these cells are present in mucous membranes and tissues. Numerous studies in mice have shown that monocytes circulate in the blood and are recruited to mucosal tissues or inflammation sites, where they can differentiate into monocyte-derived macrophages or monocyte-derived dendritic cells (Citation12). In models of inflammatory disorders, monocyte-derived cells have been shown to exert a deleterious role, in particular by fueling the inflammation and inducing tissue damage (Citation12).
Regarding the changes in healthy controls, we believe that the same explanation applies for the significantly higher count of eosinophils, CD16+ eosinophils and monocytes out of pollen season, i.e. the increased need for eosinophils during winter to fight respiratory infections.
We realize that to accurately determine the effect of biologic therapy on leukocytes and T lymphocytes in AD patients, it is necessary to evaluate these monitored parameters in one group of patients before and after initiation of biologic therapy and to monitor these parameters during the course of treatment. However, our results suggest an effect of biological treatment. We selected patients to match the age, sex, and severity of AD before starting biologic therapy, as well as the presence of other atopic diseases such as bronchial asthma and allergic rhinitis.
In conclusion, the immunological profile in patients with atopic dermatitis, but also in healthy individuals, varies throughout the year. Although there was a significant improvement in skin findings in patients treated with dupilumab, the changes in immunological profile show a persistent altered immune response.
Explanation to
The absolute and relative count of leukocytes, lymphocytes, neutrophils and monocytes in AD patients with and without dupilumab therapy in pollen season and out of pollen season compared to control group-horizontal comparison. The comparison of results in AD patients and in control group in pollen season and out of pollen season-vertical comparison. The median value is recorded. DUP – patients without dupilumab treatment, DUP + patients with dupilumab treatment. NK cells – natural killer cells. 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.
Explanation to
The absolute and relative count of eosinophils, relative CD16+ eosinophils, the absolute and relative count of basophils and relative CD203+ basophils in AD patients with and without dupilumab therapy in pollen season and out of pollen season compared to control group-horizontal comparison. The comparison of results in AD patients and in control group in pollen season and out of pollen season-vertical comparison. The median value is recorded. DUP – patients without dupilumab treatment, DUP + patients with dupilumab treatment. NK cells – natural killer cells. 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.
Explanation to
The absolute and relative count of T lymphocytes (CD4+, CD8+, regulatory T lymphocytes) and NK cells in AD patients with and without dupilumab therapy in pollen season and out of pollen season compared to control group-horizontal comparison. The comparison of results in AD patients and in control group in pollen season and out of pollen season-vertical comparison. The median value is recorded. DUP – patients without dupilumab treatment, DUP + patients with dupilumab treatment. NK cells – natural killer cells. 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.
Ethical approval
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.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Data availability statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Additional information
Funding
References
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