Dear editor,the aim of our study is to show the changes of B lymphocytes in atopic dermatitis (AD) patients and in healthy subjects in pollen season and out of pollen season. B cells are not examined as well as T cells. It is not yet known what is the count of B lymphocytes and their subsets and CD5+, CD22+ and CD73+ B lymphocytes in AD patients in pollen season and out of pollen season in AD patients with and without dupilumab therapy. B cells play important roles in immune defense, including immunoglobulin production, antigen presentation, and cytokine secretion; some data suggest that B-cell dysregulation might affect the pathogenesis of T cell–mediated diseases, such as AD (Citation1–6). The number and function of B lymphocytes can be affected by allergen exposure, including pollen. In our region, the significant role play allergens from PR 10 proteins Bet v 1 (Birch), Aln g 1 (Alder) and Cor a1.0103 (Corylus avelana) (Citation7), beta expansin (Lol p 1, Phl p 1), molecular components from Timothy and cultivated rye (Secc pollen), (Citation7). Dupilumab is a human monoclonal antibody directed against subunit α of the IL-4 receptor, which is also partf the IL-13 receptor. Through inhibition of the signaling of these two cytokines, dupilumab significantly improves symptoms of AD (Citation8).
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 (first. and third quartil 27.2–40.5) and the other group consisted of patients treated with dupilumab (13 patients − 7 men, 6 women, age 43.4 years (first. and third quartil 38.6-48.3). 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. All patients underwent a comprehensive dermatological examination. As a control group, forty healthy individuals in pollen season and thirty healthy individuals out of pollen season, all blood donors with negative total IgE, were examined at Faculty Hospital Hradec Králové, Charles University, Czech Republic. The representation of the control group matched with regard to gender and age to AD patients.
In all AD patients (with and without dupilumab therapy) included in the study, we analyzed the results of specific IgE to molecular components of pollen and other allergens (Citation7). The specific IgE was examined with the use of ALEX2 Allergy Xplorer test (Citation7).
The determination of the number of leukocytes, or lymphocytes within the leukocyte differential, was performed on Sysmex XN-10 blood cell analyzers using a combination of hydrodynamic focusing and flow fluorescence cytometry. The count of B lymphocytes and the count of CD5+, CD22+ and CD73+ B lymphocytes was evaluated with the immunophenotyping with flow cytometry. For statistical analysis we used non-parametric 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 (2021). NCSS, LLC. Kaysville, Utah, USA, ncss.com/software/ncss.
The characteristic of patients with evaluation of pollen allergy to molecular components, severity of AD and the quality of life is recorded in .
Table 1. Characteristic of patients. The characteristic of 13 AD patients with dupilumab therapy and of 21 AD patients without dupilumab therapy. The number of patients (%) with positive result to molecular components according to ALEX2 Allergy Xplorer. The mean values (minimal, maximal) 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. aage is recorded at first examination during period out of pollen season. bThe mean values (minimal, maximal) of SCORAD, EASI before starting the dupilumab therapy.
The results of laboratory examinations (the count of leukocytes, lymphocytes, B lymphocytes with their subsets, CD5+, CD22+, CD73+ B lymphocytes) are presented in Tables.
We demonstrate the results of laboratory examinations in AD patients with and without dupilumab therapy in pollen season and out of pollen season compared to control group (horizontal comparison). We compare the results of laboratory examinations between two groups of AD patients and in control subjects in pollen season and out of pollen season (vertical comparison). In we show the count of absolute leukocytes and absolute lymphocytes with statistical analysis. In we show the count of B lymphocytes with theit subsets and the count of CD5+, CD22+ and CD73+ B lymphocytes with statistical analysis.
Table 2. The count of leukocytes and lymphocytes n 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 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. We show the significant difference in statistical analysis, p-value <0.05.
Tables 3. The count of B lymphocytes and their subsets and CD5+, CD22+, CD73+ B lymphocytes in AD patients with and without dupilumab therapy in pollen season and out of pollen season compared to control group – horizontal comparison.
Our study shows interesting results:
In pollen season, the count of total memory B lymphocytes is significantly lower and the count of naive B lymphocytes is significantly higher in both controls and AD patients with and without dupilumab therapy compared to the count out of pollen season (). The count of non-switched B lymphocytes in AD patients and in control is significantly lower in pollen season, but in addition, the count of non-switched B lymphocytes in the pollen season is significantly higher in dupilumab patients than in the control group.
When comparing the results in pollen season and out of pollen season with respect to the control group, we demonstrated these significant changes: in pollen season we show the higher count of non-switched B lymphocytes in dupilumab patients, the higher count of transitional B lymphocytes in patients withhout dupilumab compared to controls. Out of pollen season we show the lower count of relative CD22 B lymphocytes in both groups of AD patients compared to controls ().
Although we demonstrated other significant statistical differences in the count of B lymphocytes between pollen season and out of pollen season in AD patients, we consider them to be changes within normal limits because their values are not significantly different from those of the control group. These changes include an increase of relative B lymphocytes in AD patients without dupilumab in pollen season; in both groups of AD patients we observe a decrease in switched B lymphocytes in the pollen season, a rise in transitional B lymphocytes in AD patients with dupilumab therapy, and a rise in CD5+ (absolute and relative) and CD73+ (relative) B lymphocytes, . Despite the significant differences in the pollen season and out of pollen season, these parameters are within normal limits in these AD patients compared to the control group.
In conclusion, it is the first study demonstrating the changes in the count of B lymphocytes in different seasons in the year in AD patients suffering from moderate and severe form compared to control subjects. In addition, we also evaluate what changes occur in patients treated with dupilumab. Our study shows, that normalization of the count of transitional lymphocytes occurs in patients treated with dupilumab in pollen season; in patients without dupilumab we observe significantly higher count of transitional lymphocytes during pollen season. On the other hand we show the higher count of non-switched B lymphocytes in AD patients treated with dupilumab during pollen season, which is not observed in AD patients without dupilumab. Significant changes occur also in healthy subjects in pollen season in the count of memory, naive and non-switched B lymphocytes.
Authors contribution
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.
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.The study was conducted in accordance with the Helsinki Declaration of 1964 and all subsequent amendments, and all patients provided written informed consent. Patient-level data used for this analysis were de-identified.
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.
Informed Consent
Informed consent was obtained from all subjects involved in the study.
Disclosures statement
No potential conflict of interest was reported by the author(s). Jarmila Čelakovská, Eva Čermákova, Petra Boudková, Ctirad Andrýs, Jan Krejsek has nothing to disclose.
Data availability [database]
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Additional information
Funding
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