TYPE Original Research PUBLISHED 13 March 2023 DOI 10.3389/fimmu.2023.1124018 Allergen sensitization stratifies OPEN ACCESS IL-31 production by memory T EDITED BY Jongdae Lee, Guangzhou Medical University, China cells in atopic dermatitis patients REVIEWED BY Hans David Brightbill, Lı́dia Sans-de San Nicolàs 1, Ignasi Figueras-Nart 2, Genentech Inc., United States Patrick M. Brunner, Irene Garcı́a-Jiménez 1, Montserrat Bonfill-Ortı́ 2, Icahn School of Medicine at Mount Sinai, United States Antonio Guilabert 3, Laia Curto-Barredo 4, *CORRESPONDENCE Marta Bertolı́n-Colilla 4, Marta Ferran 4, Esther Serra-Baldrich 5, Luis F. Santamaria-Babı´ Ramon M. Pujol 4 and Luis F. Santamaria-Babı́ 1* luis.santamaria@ub.edu 1 Immunologia Translacional, Departament de Biologia Cel•lular, Fisiologia i Immunologia, Facultat de SPECIALTY SECTION Biologia, Universitat de Barcelona (UB), Parc Científic de Barcelona (PCB), Barcelona, Spain, This article was submitted to 2 Departament de Dermatologia, Hospital de Bellvitge, Universitat de Barcelona (UB), L’Hospitalet de T Cell Biology, Llobregat, Spain, 3 Departament de Dermatologia, Hospital General de Granollers, Granollers, Spain, a section of the journal 4 Departament de Dermatologia, Hospital del Mar, Institut Hospital del Mar d’Investigacions Mèdiques Frontiers in Immunology (IMIM), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain, 5 Departament de Dermatologia, RECEIVED 14 December 2022 Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain ACCEPTED 28 February 2023 PUBLISHED 13 March 2023 CITATION Sans-de San Nicolàs L, Figueras-Nart I, Background: The role of allergen sensitization in IL-31 production by T cells and Garcı´a-Jiménez I, Bonfill-Ortı´ M, specifically in the clinical context of atopic dermatitis (AD) has not Guilabert A, Curto-Barredo L, Bertolı´n-Colilla M, Ferran M, been characterized. Serra-Baldrich E, Pujol RM and Santamaria-Babı´ LF (2023) Allergen Methods: The response to house dust mite (HDM) in purified memory T cells sensitization stratifies IL-31 production by memory T cells in atopic cocultured with epidermal cells from AD patients (n=58) and control subjects dermatitis patients. (n=11) was evaluated. AD-associated cytokines from culture supernatants, Front. Immunol. 14:1124018. doi: 10.3389/fimmu.2023.1124018 plasma proteins and mRNA expression from cutaneous lesions were assessed and related with the clinical features of the patients. COPYRIGHT © 2023 Sans-de San Nicolàs, Figueras-Nart, Garcı´a-Jiménez, Bonfill-Ortı´, Guilabert, Results: HDM-induced IL-31 production by memory T cells defined two subsets Curto-Barredo, Bertolı´n-Colilla, Ferran, of AD patients according to the presence or absence of IL-31 response. Patients Serra-Baldrich, Pujol and Santamaria-Babı´. This is an open-access article distributed in the IL-31 producing group showed a more inflammatory profile, and increased under the terms of the Creative Commons HDM-specific (sp) and total IgE levels compared to the IL-31 non-producing Attribution License (CC BY). The use, distribution or reproduction in other group. A correlation between IL-31 production and patient’s pruritus intensity, forums is permitted, provided the original plasma CCL27 and periostin was detected. When the same patients were author(s) and the copyright owner(s) are analyzed based on sp IgE and total IgE levels, an increased IL-31 in vitro credited and that the original publication in this journal is cited, in accordance with response, as well as type 2 markers in plasma and cutaneous lesions, was accepted academic practice. No use, found in patients with sp IgE levels > 100 kUA/L and total IgE levels > 1000 kU/ distribution or reproduction is permitted L. The IL-31 response by memory T cells was restricted to the cutaneous which does not comply with these terms. lymphocyte-associated antigen (CLA)+ T-cell subset. Frontiers in Immunology 01 frontiersin.org Sans-de San Nicolàs et al. 10.3389/fimmu.2023.1124018 Conclusion: IgE sensitization to HDM allows stratifying IL-31 production by memory T cells in AD patients and relating it to particular clinical phenotypes of the disease. KEYWORDS atopic dermatitis, CCL27, CLA+ memory T cells, house dust mite, IgE, IL-31, periostin, pruritus 1 Introduction production by memory T cells compared to patients with lower sp and total IgE levels. These results show for the first time a IL-31 is a clinically relevant neuroimmune cytokine involved in relationship between allergen-specific T-cell-mediated IL-31 epidermal barrier disruption, pruritus, inflammation and tissue production, clinical status of the patients and the levels of remodeling in atopic dermatitis (AD) (1–3). It is enhanced in the allergen sensitization that can be of help to guide patient’s sera from AD patients and correlates with disease severity (4). identification in response to IL-31-directed therapies. The major source of IL-31 are CD4+ T cells associated to a Th2 phenotype (5–8), but it is also produced by dendritic cells, mast cells, group 2 innate lymphoid cells, basophils, eosinophils or M2 2 Materials and methods macrophages (9–14). Its receptor, IL-31RA, is expressed by multiple cell types, including skin sensory neurons, keratinocytes and 2.1 Patients immune cells, activating the JAK/STAT, PI3K/AKT and MAPK/ JNK pathways (9, 15, 16). Peripheral blood and two skin biopsies from active lesional IL-31 and IL-31RA expression is increased in the AD skin and areas were collected from 58 consented moderate-to-severe AD are part of the “core” signature characterized by the activation of the patients and 11 consented controls under institutional review IL-31/IL-1 signaling pathway (17). The epidermal barrier function board-approved protocols at the Hospital de Bellvitge, Hospital is affected by IL-31 modulation of keratinocyte differentiation General de Granollers, Hospital del Mar and Hospital de la Santa directly or via other mediators including IL-20, IL-24, IL-33 and Creu I Sant Pau (Spain). Exclusion criteria included topical or IL-1a (18–23). IL-31 binding to its receptor in sensory neurons systemic anti-inflammatories for the last 2 or 4 weeks prior to the stimulates itch signaling, neuronal branching and increases of study, respectively. cutaneous nerve fiber density (9, 24). A clear improvement of Plasma samples were assessed for total IgE (kU/L) and HDM- pruritus has been observed after IL-31RA blockade in moderate- specific IgE (response (OD) and kUA/L) by ImmunoCAP (Thermo to-severe AD patients (25–27). IL-31 initiates and maintains Fisher Scientific, Waltham, MA, USA). Serum samples were used for inflammation by promoting the release of proinflammatory lactate dehydrogenase (LDH) measurement (U/L) in a diagnostic cytokines and chemokines by myeloid cells, eosinophils, basophils laboratory. Patients’ characteristics are summarized in Table S1. and keratinocytes (10, 15, 18–20, 28–31). Aeroallergens such as house dust mite (HDM) are associated to disease severity and total IgE levels in AD (32, 33). The relationship 2.2 Isolation of memory CLA+/- T cells and between IL-31 and allergens has been poorly characterized and epidermal cells suspension mainly limited to canine models: HDM-sensitized dogs showed IL- 31 expression in CD3+ CD4+ T cells in the skin lesions and Central and effector memory CD45RA- T lymphocytes were production of IL-31 by Th2-polarized peripheral blood purified from PBMC after Ficoll (GE Healthcare, Princeton, NJ, mononuclear cells (PBMC) stimulated with HDM (7, 34). Non- USA) gradient and three consecutive immunomagnetic separations lesional skin from AD patients challenged with HDM induced IL-31 (Miltenyi Biotech, Bergisch Gladbach, Germany) (36). First CD14+ mRNA expression in three out of six HDM allergic patients (21). and CD19+ cells were depleted, then CD16+ and CD45RA+ Increased frequencies of IL-31+ CD4+ and CD8+ T cells in PBMC lymphocytes were depleted, and finally CD45RA- T cells were from HDM-sensitized patients have also been detected (35). divided into CLA+ and CLA- T-cell subpopulations using the Nevertheless, no studies have addressed the role of allergen in IL- Miltenyi product ref 130-092-464. Sample purity was >95% of 31 production by memory T cells and its possible relationship with CLA-positivity for the CLA+ T-cell subpopulation and <10% of particular clinical features in AD patients. CLA-positivity for the CLA- T-cell subpopulation (36). Terminal In this study, HDM-induced IL-31 by cutaneous lymphocyte- differentiated memory TEMRA cells were not studied with this associated antigen (CLA)+ memory T cells correlated with patient’s approach because of CD45RA+ cells depletion. pruritus. Patients with elevated HDM-specific (sp) IgE (> 100 kUA/ Punch skin biopsy samples were incubated in dispase solution L) and total IgE levels (> 1000 kU/L) showed an increased IL-31 (Corning, Corning, NY, USA) overnight at 4°C, and the epidermal Frontiers in Immunology 02 frontiersin.org Sans-de San Nicolàs et al. 10.3389/fimmu.2023.1124018 sheet was peeled off from the dermis. The epidermis was cut into instrument (Invitrogen) and analysed with ProcartaPlex Analyst pieces and incubated with trypsin (Biological Industries, Kibbutz software version 1.0 (Invitrogen). Values below the lower limit of Beit Haemek, Israel) for 15 minutes at 37°C. Then, the epidermal quantification (LLOQ) were treated as zero. Plasma CCL22 levels tissue was mechanically disaggregated by pipetting and the cell were quantified with ProcartaPlex. suspension was transferred to fresh culture media (1:1 volume) (RPMI supplemented with 10% FBS (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA)). 2.6 ELISA Finally, the epidermal cells suspension (Epi) was obtained by means of centrifugation. It contained all cell types at the same proportion Pre-coated ELISA kits were used for quantification of plasma present in the epidermis, being keratinocytes the major cell type. levels of CCL27, CCL17, CCL18, sIL-2R (Invitrogen) and periostin (AdipoGen Life Sciences, San Diego, CA, USA). 2.3 Coculture of CLA+/- memory T cells with epidermal cells and stimulation 2.7 RNA isolation, quantitative real-time PCR and gene-array analysis The coculture system was performed by seeding 3 x 104 autologous epidermal cells with 5 x 104 circulating CLA+ or CLA- Lesional skin biopsy specimens frozen in Tissue-Tek O.C.T. memory T cells (CLA+/Epi or CLA-/Epi, respectively) in a 96-well Compound (Sakura Finetek, Alphen aan den Rijn, The U-bottom plate (Falcon, Corning, Corning, NY, USA), in the Netherlands) at -80°C were used for RNA isolation by using the culture media described above. Whole (CD4+ and CD8+) CLA+ TRIzol Reagent (Invitrogen). memory T cells were used; however, we assume that results For qRT-PCR, cDNA was obtained with the High Capacity obtained are due to CD4 + CLA + memory T cells, since the cDNA Reverse Transcription kit (Applied Biosystems, Waltham, majority of CLA+ memory T cells are CD4+ (80% CD4+ and 20% MA, USA) and preamplified with the TaqMan PreAmp Master Mix CD8+) and IL-31 is mainly produced by CD4+CLA+ T cells (6, 23). (2x) (Applied Biosystems). Taqman Gene Expression Master Mix Cocultures were left untreated (M) or activated for 5 days with and FAM-labelled probes (Table S2) (Applied Biosystems) were HDM extract kindly provided by LETI Pharma at 10 µg/mL or 24 used for qRT-PCR with an ABI Prism 7900HT instrument (Applied hours with staphylococcal enterotoxin B (SEB) (Sigma-Aldrich) at Biosystems). Data was processed by SDS analysis software version 100 ng/mL final well concentration (37). In cultures containing only 2.4.1 (Applied Biosystems) and gene expression was calculated by T cells or epidermal cells, the mentioned amounts of each cell type using the D - D cycle threshold (Ct) method (with the mean cycle were used, and activation with HDM was performed in the same threshold value for RPLP0 and the gene of interest for each sample). way. Collected supernatants were kept at -20°C. The equation 1.8e (CtRPLP0 - Ctgene of interest) x 104 was used for For blocking assays, HLA-A/B/C (class I) (BioLegend, San normalizing the values (38). Diego, CA, USA), HLA-DR (class II) (BioLegend), CD1a (Bio X For gene-array analysis, quality control of concentration and Cell, Lebanon, NH, USA), IL-33 (R & D Systems, Minneapolis, integrity of the isolated RNA was performed with the NanoDrop Minnesota, USA) neutralizing antibodies, or respective mouse One (Thermo Fisher Scientific) and the Agilent 2100 Bioanalyzer IgG2a isotype control (BioLegend), mouse IgG1 (BioLegend) and (Agilent Technologies, Santa Clara, CA, USA). PrimeView Human goat IgG isotype control (R & D Systems) were added to cocultures Gene Expression Arrays (Applied Biosystems) were processed at the at 10 µg/mL (for HLA and CD1a blocking assays) or 1 µg/mL (for Functional Genomics Facility of IRB Barcelona (Barcelona, Spain) IL-33 blocking assay) final concentration. and raw data (CEL files) were processed with Transcriptome Analysis Console version 4.0 (Applied Biosystems). Raw data 2.4 Transwell culture were deposited in the Gene Expression Omnibus repository, accession number GSE226073. Genes with a fold change (FCH) 5 x 104 purified CLA+ or CLA- memory T cells were added to of 1.5 or greater and a p value of less than .05 were considered the top chamber of 0.4 µm pore size polycarbonate 96-well HTS differentially expressed genes (DEG). Pathway enrichment analysis Transwell culture insert (Corning) containing 3 x 104 autologous was performed using g:Profiler web server (https://biit.cs.ut.ee/ lesional epidermal cells stimulated with HDM at 10 µg/mL final well gprofiler/gost) with Gene Ontology (GO) biological process concentration in the bottom chamber. Supernatants were collected database and a Benjamini-Hochberg FDR of .05 or greater. at day 5 of culture. 2.8 Statistical analyses 2.5 Cytokine and chemokine quantification Data analysis and representation were performed with GraphPad ProcartaPlex immunoassay (Invitrogen, Waltham, MA, USA) Prism software version 8 (GraphPad Software Corporation, San was used to measure IL-31, IL-13, IL-4, IL-5, IL-17A, IL-22 and Diego, CA, USA). Data are represented as the median ± 95% IFN-g concentrations in the culture supernatants with the MAGPIX confidence interval (CI). Wilcoxon test was used to compare two Frontiers in Immunology 03 frontiersin.org Sans-de San Nicolàs et al. 10.3389/fimmu.2023.1124018 conditions within the same group and Mann-Whitney test was used from group 1 showed increased production of IL-13, IL-4, IL-5, IL- to compare two groups. In tables, sample median (25th-75th 17A and IL-22 compared to group 2 (Figures 2A; S4B), revealing a percentiles) and Mann-Whitney test were used for continuous more inflammatory phenotype in group 1 versus group 2. Also, in variables, raw numbers (percentages) and Fisher’s exact test were group 1 IL-31 production directly correlated with IL-13 (r = 0.62, p used for categorical variables, and bold values indicate significant = .0093) and IL-4 (r = 0.85, p <.0001), but no correlation with IL-5, data. Correlations were examined using Spearman coefficient and IL-17A, IL-22 and IFN-g was observed (Figure 2B). Although group represented with linear regression. Differences were considered 2 of patients were defined for their null production of IL-31 by significant at a P-value of less than .05 and showed as: (*) p <.05; HDM-stimulated CLA+ T cells, there was an heterogeneous IL-13, (**) p <.01; (***) p <.001; (****) p <.0001. IL-4, IL-5, IL-17A, IL-22 and IFN-g response in the CLA+ T-cell cocultures of this group (Figure S5). All correlations were tested with and without outlier patient data. 3 Results When stimulating cocultures with Staphylococcus aureus superantigen B (SEB), another clinically relevant stimulus of AD, 3.1 HDM induces IL-31 in memory T cells IL-31 was specifically produced by CLA+ T cells but no significant cultured with autologous lesional differences were found between group 1 and group 2 of patients epidermal atopic dermatitis cells, which (Figure S6A). Additionally, therewas no correlation between HDM- correlates with pruritus, and plasma CCL27 and SEB-induced IL-31 production by memory T cells (Figure S6B). and periostin AD-derived cocultures containing circulating memory CLA+ T cells and autologous lesional epidermal cells (CLA+/Epi) in the 3.2 Patients with memory T-cell IL-31 presence of HDM led to IL-31 production, whereas CLA- T-cell AD response to HDM show elevated plasma cocultures (CLA-/Epi) and control (C)-derived cocultures did not levels of sp and total IgE and a more produced IL-31 upon stimulation (Figure 1A). Epidermal cells inflammatory profile than patients with no suspension promoted HDM-induced CLA+ T-cell-derived IL-31 IL-31 response response (Figure S1A) and it was produced in a time-dependent manner (Figure S1B). Patients from group 1 displayed increased sp IgE and total IgE AD patients were classified into two groups to further levels compared with patients from group 2 (Table 1). No characterize clinical profiles based on the differential response to differences were found for severity, pruritus, eosinophilia, years HDM: those producing IL-31 by CLA+ T cells were arranged into since diagnosis and comorbidities. Although group 2 of patients had group 1 (n=17), and those with no IL-31 production were arranged a reduced allergen sensitization degree when compared to group 1 into group 2 (n=41) (Figure 1A). of patients, they showed higher total IgE and sp IgE levels than Within group 1 of patients, HDM-induced IL-31 response controls (total IgE (kU/L): group 2 median = 568.00, C median = directly correlated with pruritus (r = 0.67, p = .0036; Figure 1B) 21.10, p <.0001; sp IgE response (OD): group 2 median = 5338, C and plasma levels of CCL27 (r = 0.62, p = .0090; Figure 1C) and median = 27.00, p <.0001). periostin (r = 0.56, p = .050; Figure 1D). Correlations within group 2 To further characterize both groups, we evaluated changes in of patients could not be performed due to undetectable IL-31 lesional skin tissue from patients belonging to group 1 and group 2 production in the cocultures. As shown in Figure 1E, there was using gene-array analysis. We identified two hundred six probe-sets direct correlation between plasma CCL27 levels and SCORAD in (143 unique genes) up-regulated and one hundred probe-sets (80 group 1 (r = 0.56, p = .032) but not in group 2 (r = 0.31, p = .091). gens) down-regulated in group 1 skin compared with group 2 skin Additionally, HDM-induced CLA+ T-cell IL-31 nearly correlated (Figure S7A; Table S3). Among the upregulated genes there were IL- with SCORAD in group 1 of patients (r = 0.49, p = .066; not shown). 20, IL-24, CCL20, CXCL1, CCL2 and EGR1. Only for IL-20 was There was no correlation between IL-31 in vitro response to HDM FDR of less than .05. Enrichment tests of DEG revealed that group 1 and plasma levels of HDM-specific (sp) IgE and total IgE, although lesional skin was enriched for biological processes related to the there was a tendency for the former (r = 0.46, p = .068; Figure S2). immune system such as response to external stimulus, The IL-31 production by CLA+ memory T cells was blocked by inflammatory response and cellular response to chemokine 59% by a neutralizing HLA class II, but not HLA class I, antibody (Figure S7B; Table S4). On the contrary, group 2 lesional skin (Figures 1F; S3A). Blocking of IL-33 and CD1a molecules did not was enriched for developmental processes (Figure S7C; Table S5). affect IL-31 production (Figures S3B, C), but cell-cell contact Interestingly, difference in IL-20 expression between group 1 between CLA+ T cells and epidermal cells was necessary for IL-31 and group 2 of patients by gene-array analysis was confirmed by production as demonstrated by transwell cultures (Figure 1G). qRT-PCR (Figure S7D), but higher number of patients should be IL-31 was simultaneously measured with other AD-derived analyzed to confirm this data due to significant overlap between mediators (IL-13, IL-4, IL-5, IL-17A, IL-22 and IFN-g) and we both groups. Additionally, increase in IL-17A and IL-21 mRNA found that IL-13, IL-4, IL-5, IL-17A and IL-22 production by expression was detected in group 1 compared to group 2 (Figure HDM-induced CLA+ T-cell AD cocultures was higher than that S7D), and no differences were found for IL-31 mRNA expression in by C cocultures (Figure S4A). HDM-activated CLA+/Epi cocultures cutaneous lesions between both groups. Frontiers in Immunology 04 frontiersin.org Sans-de San Nicolàs et al. 10.3389/fimmu.2023.1124018 A B C D E F G FIGURE 1 HDM-induced IL-31 by memory T cells correlates with patient’s pruritus and plasma CCL27 and periostin, partially depends on HLA class II molecules and the cell-cell contact with epidermal cells, and is restricted to CLA+ T-cells subset. (A) IL-31 (pg/mL) produced by CLA+/- T-cell cocultures in basal conditions (M) or stimulated with HDM in AD-(n=58) and C-(n=11) derived samples. Correlations of CLA+/Epi/HDM IL-31 production in group 1 with (B) pruritus (n=17), and plasma (C) CCL27 (n=17) and (D) periostin (n=13) levels. (E) Correlation between plasma CCL27 levels and SCORAD in group 1 (n=15) and group 2 (n=31) of patients. (F) HDM-activated CLA+/- T-cell cocultures were treated with HLA class II neutralizing antibody or control IgG isotype at day 0, and IL-31 production (pg/mL) at day 5 was compared with respect to isotype values (n=6). Red bar indicates the mean. (G) CLA+/- T cells were added in the upper chamber and HDM-stimulated epidermal cells suspension in the lower chamber of the 96-well transwell plate and IL-31 (pg/mL) was measured at day 5. Data for three representative patients. AD, atopic dermatitis; C, control subjects; CLA, cutaneous lymphocyte-associated antigen T cells; Epi, epidermal cells suspension; HDM, house dust mite; M, untreated; SCORAD, scoring atopic dermatitis. ns: p >.05; *p <.05; **p <.01; ****p <.0001. Frontiers in Immunology 05 frontiersin.org Sans-de San Nicolàs et al. 10.3389/fimmu.2023.1124018 A B FIGURE 2 Patients producing IL-31 (group 1) by HDM-stimulated CLA+ T cells have a more inflammatory phenotype than patients with undetectable IL-31 (group 2). (A) IL-13, IL-4, IL-5, IL-17A, IL-22 and IFN-g (pg/mL) produced by HDM-induced CLA+ T-cell cocultures was compared between groups 1 (n=13-17) and 2 (n=35-41) of patients. (B) In group 1, HDM-stimulated CLA+ T-cell-dependent IL-31 production was correlated with IL-13 (n=17), IL- 4 (n=17), IL-5 (n=17), IL-17A (n=17), IL-22 (n=13) and IFN-g (n=17). CLA, cutaneous lymphocyte-associated antigen T cells; Epi, epidermal cells suspension; HDM, house dust mite. ns: p >.05; **p <.01; ***p <.001; ****p <.0001. Frontiers in Immunology 06 frontiersin.org Sans-de San Nicolàs et al. 10.3389/fimmu.2023.1124018 TABLE 1 Differences in clinical characteristics between group 1 and group 2. Group 1 Group 2 r Clinical data Median (25-75% percentiles) or Total N Median (25-75% percentiles) or Total N Value number (%) number (%) Age 28.00 (18.50-44.50) 17 36.00 (27.00-45.75) 40 .063 SCORAD 59.63 (30.00-61.32) 15 59.27 (42.10-68.00) 31 .44 EASI 22.65 (19.38-27.93) 16 25.00 (15.25-32.00) 38 .92 IGA 4.00 (3.00-4.00) 16 3.00 (3.00-4.00) 40 .35 Pruritus (VAS) 7.00 (6.50-8.00) 17 8.00 (6.25-9.00) 40 .31 3 Eosinophilia (10 /uL) 0.73 (0.21-1.06) 16 0.46 (0.24-0.61) 34 .16 sp IgE response (OD) 18747 (12287-20456) 17 5338 (393.0-16830) 41 <.0001 Total IgE (kU/L) 2000 (606.9-5000) 17 568.0 (110.5-1792) 41 .0027 LDH (U/L) 209.0 (143.8-249.8) 6 193.0 (128.0-236.3) 26 .66 Years since AD 15.50 (11.00-25.75) 16 24.00 (13.00-33.50) 38 .28 diagnosis Comorbidities, n 13 (81.25) 16 27 (67.50) 40 .35 Rhinitis, n 8 (50.00) 16 23 (58.97) 39 .56 Asthma, n 6 (37.50) 16 16 (41.03) 39 >.99 Conjunctivitis, n 8 (50.00) 16 15 (38.46) 39 .55 Food allergy, n 2 (12.50) 16 11 (29.73) 37 .30 Categorical variables are presented as counts (percentages) and continuous variables are presented as medians (25th-75th percentiles). AD, atopic dermatitis; EASI, eczema area and severity index; IGA, investigator’s global assessment; LDH, lactate dehydrogenase; OD, optical density; SCORAD, scoring atopic dermatitis; VAS, visual analogue scale. Bold values indicate significant data. 3.3 Patients with sp IgE > 100 kUA/L have increased IL-31 in vitro response to HDM A B confined to the CLA+ memory T-cell subset compared to patients with sp IgE < 100 kUA/L An alternative analysis in the same patients revealed that those with high levels of sp IgE (> 100 kUA/L) showed increased HDM-induced IL-31 response by memory T cells, only in the CLA+ T-cell subset, than those with low levels of sp IgE (< 100 kUA/L) (Figure 3A). There was a trend towards positive correlation between IL-31 response by HDM- C stimulated CLA+ T-cell cocultures and SCORAD in the high sp IgE group (r = 0.28, p = .28), whereas this trend was negative for the low sp IgE group (r = -0.32, p = .094; Figure 3B). Patients from the high sp IgE group showed increased eosinophilia and total IgE levels compared to patients from the low sp IgE group, as expected (Table 2). Additionally, in the high sp IgE there was a tendency towards more patients with FIGURE 3 conjunctivitis, a sign of HDM clinical symptom (p <.1). Enhanced IL-31 in vitro response and plasma levels of CCL18 and CCL22 in patients with sp IgE > 100 kUA/L. (A) IL-31 (pg/mL) from In line with the previous observation, the IL-31 mRNA AD culture supernatants separated according to sp IgE levels (> 100 expression in cutaneous lesions was greater in the high than in kUA/L n=21, < 100 kUA/L n=37). (B) Correlation between CLA+/Epi/ the low sp IgE groups (Figure S8A). Furthermore, plasma levels of HDM IL-31 and SCORAD in high (n=17) and low (n=29) sp IgE groups. (C) Plasma levels of CCL18 (ng/mL) and CCL22 (pg/mL) CCL18, CCL22, CCL27, periostin and sIL-2R were elevated in the were compared between high (n=16-21) and low (n=30-37) sp IgE high compared to the low sp IgE groups (Figures 3C; S8B). groups. CLA, cutaneous lymphocyte-associated antigen T cells; Epi, epidermal cells suspension; HDM, house dust mite; M, untreated; sp Although results were significant (p <.05) there was clear overlap IgE, specific IgE to house dust mite; SCORAD, scoring atopic between both groups and increased number of patients would dermatitis. ns: p >.05; *p <.05; **p <.01; ***p <.001. be required. Frontiers in Immunology 07 frontiersin.org Sans-de San Nicolàs et al. 10.3389/fimmu.2023.1124018 TABLE 2 Differences in clinical characteristics between patients with sp IgE >100 kUA/L and patients with sp IgE <100 kUA/L. Sp IgE >100 kUA/L Sp IgE <100 kUA/L r Clinical data Median (25-75% percentiles) or Total N Median (25-75% percentiles) or Total N Value number (%) number (%) Age 36.00 (23.50-48.00) 21 34.50 (26.25-45.00) 36 .85 SCORAD 59.63 (51.00-62.76) 17 56.00 (37.50-66.58) 29 .52 EASI 27.30 (19.25-34.75) 20 22.10 (11.90-27.41) 34 .13 IGA 4.00 (3.00-4.00) 20 3.00 (3.00-4.00) 36 .21 Pruritus (VAS) 7.00 (6.50-8.00) 21 8.00 (6.25-9.00) 36 .40 3 Eosinophilia (10 /uL) 0.74 (0.47-1.08) 19 0.33 (0.19-0.56) 31 .0026 sp IgE response (OD) 19507 (17643-20456) 21 4031 (312.5-10513) 37 <.0001 Total IgE (kU/L) 3639 (1962-5000) 21 336.0 (107.0-1130) 37 <.0001 LDH (U/L) 237.0 (124.5-329.3) 8 184.5 (122.0-219.3) 24 .13 Years since AD 18.00 (13.50-32.75) 20 23.50 (9.50-30.00) 34 .99 diagnosis Comorbidities, n 17 (80.95) 21 23 (65.71) 35 .36 Rhinitis, n 13 (61.90) 21 18 (52.94) 34 .58 Asthma, n 7 (33.33) 21 15 (44.12) 34 .57 Conjunctivitis, n 12 (57.14) 21 11 (32.35) 34 .094 Food allergy, n 2 (9.52) 21 11 (34.38) 32 .053 Categorical variables are presented as counts (percentages) and continuous variables are presented as medians (25th-75th percentiles). AD, atopic dermatitis; EASI, eczema area and severity index; IGA, investigator’s global assessment; LDH, lactate dehydrogenase; OD, optical density; SCORAD, scoring atopic dermatitis; VAS, visual analogue scale. Bold values indicate significant data. The IL-31 response by memory T cells activated with SEB levels of CCL27 and periostin. In addition, plasma levels of HDM- showed no significant differences between high sp IgE and low sp specific IgE and total IgE were increased in group1. IgE groups (Figure S8C). Additionally, no correlation between the IL-31 is one of the main drivers of pruritus (2). For the first HDM- and SEB-induced IL-31 production was observed in the high time, a direct correlation between memory T-cell-derived IL-31 sp IgE and the low sp IgE groups (Figure S8D). response to HDM in vitro and patient’s pruritus was demonstrated, which underlines the clinical relevance of this mechanism in AD. This translational observation stablishes a relationship between 4 Discussion allergen and patient’s pruritus through a mechanism restricted to a subset of circulating skin-homing memory T cells. The lack of In moderate-to-severe AD the role of allergen in T-cell difference in pruritus intensity between group 1 and group 2 of mediated IL-31 production and its possible relationship with the patients may be due to the broad range of pruritogens in AD, such clinical status of the patients is presently uncharacterized. Our as IL-13 or IL-4 (1), which could promote the itch signaling results suggest that the degree of allergen sensitization allows pathway in patients that do not produce IL-31 upon HDM stratifying patients for IL-31 production by T cells with different stimulation. Since IL-31RA blockade improves symptoms in AD clinical features. (25, 27, 42), further studies should explore the influence of allergen In lesional AD skin CD4+ T cells are the most abundant sensitization and the clinical response to anti-IL-31RA therapies infiltrating lymphocytes and they are mainly CLA+ T cells (39, in pruritus. 40). Until now, T-cell-derived IL-31 production has been mainly In group 1 of patients, IL-31 produced by CLA+ T cells analyzed by qRT-PCR and intracellular flow cytometry of positively correlates with CCL27, being CLA+ T cells and CCL27 polyclonal-activated PBMC in a low number of patients and mechanistically closely related, since CLA+ T cells express the without any association with the clinic (8, 21, 23, 35, 41). In our CCR10 receptor that specifically binds CCL27 (43, 44). The study, HDM-induced IL-31 production by memory T cells defined relationship between IL-31 producers and plasma CCL27 suggests two groups of AD patients: the IL-31 producing group (group 1) that lesional keratinocytes produce the CLA-attracting chemokine and the IL-31 non-producing group (group 2). In group 1, CCL27 to facilitate further recruitment of CLA+ T cells to the compared with group 2, HDM-induced IL-31 in CLA+ T cells inflamed skin where, upon activation with HDM, would produce positively correlated with patient´s pruritus intensity, and plasma IL-31. CCL27 in the stratum corneum has been proposed as a Frontiers in Immunology 08 frontiersin.org Sans-de San Nicolàs et al. 10.3389/fimmu.2023.1124018 biomarker of clinical response to nemolizumab, an anti-IL-31RA, A recent research described an increased IL-31RA expression treatment in AD (42), supporting the significant correlation on memory B cells from AD patients with total IgE levels > 1000 between CCL27 and SCORAD in group 1 of patients. Besides kU/L compared to controls, pointing to an association between IL- this, not all patients respond to nemolizumab treatment, 31-mediated mechanisms and atopic IgE-producing phenotype suggesting that allergen sensitization may be of help to identify (56). For this reason, we classified patients according to their total anti-IL-31RA responder patients. IgE levels into high (> 1000 kU/L) and low (< 1000 kU/L) groups Periostin is an extracellular matrix protein closely related to Th2 and found an increased HDM-induced memory T-cell IL-31, IL-13 immune response with an emerging role in pruritus and barrier and IL-4 response in the former group (Figure S9A), along with dysfunction (45–48). In canine models, epicutaneous application of enhanced CCL26 mRNA expression in cutaneous lesions (Figure HDM to sensitized dogs induced upregulation of Th2 signature, S9B) and plasma levels of CCL27, periostin, CCL17, CCL18, CCL22 including IL-31 and periostin (49). Nevertheless, the association and sIL-2R (Figure S9C). These data suggest that in allergen between periostin plasma levels and IL-31, for HDM-triggered sensitized patients, IL-31 may play a role not only as T-cell- memory T cells, has not previously been reported. derived mediator by CLA+ T cells but also influencing IL-31RA- Group 1 of patients showed increased levels of IL-13, IL-4, IL-5, IL- expressing B cells. This hypothesis is currently being investigated. 17A and IL-22, all of them commonly found in AD lesional skin (17), The knowledge generated from the present study is in response to HDM by the CLA+ T-cell cocultures, suggesting a strong translationally relevant, since the allergen-induced IL-31 inflammatory response in this group. Moreover, the direct correlation production by memory T cells was confined to the CLA+ T-cell of IL-31 with IL-13 and IL-4 underlines the allergen-specific and Th2- subset, which reflects the skin-related mechanisms taking place in phenotype of IL-31, and it is also supported by the IL-4-dependent IL- AD lesions and other inflammatory cutaneous diseases (57–61). 31 production reported in polyclonal stimulated CD4+ T-cell clones Interestingly it has recently been reported that an early-term effect (5). The skin transcriptome confirmed the inflammatory signature in of dupilumab treatment on IL-13, IL-4, IL-5 and IL-22 expression is group 1 of patients, enriched in functions such as “response to external preferentially found in circulating CD4+ CLA+ T cells from AD stimulus”, “inflammatory response”, “cellular response to chemokines” patients (62, 63). Besides this, circulating allergen-specific and and “response to IL-1”. Among the up-regulated genes in this group, CLA+ T cells share the same TCRB CDR3 regions as lesional T IL-20 is associated with IL-31-induced barrier disruption (18), and has cells from AD skin (64), supporting the relevance of our results for also been related with pruritus in murine AD models (50). On the IL-31 produced by peripheral skin-homing T cells. contrary, group 2 of patients lacked a transcriptomic dysregulation of Circulating CLA+ T cells have been described to be the main inflammatory genes. memory T-cell subset producing IL-31 (23). When we Group 2 of patients responded in most of the endpoints evaluated complemented the analysis of T-cell mediated IL-31 response in in this study (e.g. severity, pruritus), which is given by the nature that the same patients by using SEB (65), IL-31 levels produced by CLA+ they are AD patients, so they share some clinical characteristics with memory T cells were lower than those observed after HDM group 1 of patients. The most significant clinical difference between stimulation, without significant differences between groups 1 and both groups was the degree of IgE sensitization to HDM, along with 2 of patients or sp IgE levels. Additionally, a correlation between total IgE levels, which were related with the differential IL-31 HDM- and SEB-induced IL-31 response was not detected, response by skin-derived memory T cells upon stimulation with suggesting different roles of these stimuli in T-cell-mediated IL-31 allergen. Thereby, two groups that apparently would be the same production in AD. The molecular mechanisms by which SEB and population may show distinct molecular mechanisms driving HDM activate T cells are different. SEB directly binds to HLA class disease immunopathogenesis. II molecules on the surface of antigen presenting cells and AD is associated with elevated IgE, sensitization to aeroallergens stimulates T cells expressing particular TCR. SEB can also induce and eosinophilia (51). Total IgE levels correlate with patients’ severity skin inflammation by the following mechanisms: the direct binding and are increased in patients sensitized to aeroallergens (33, 52). IgE to antigen presenting cells in the skin, direct binding to MHC class levels are not a diagnostic requirement, but they are useful for II keratinocytes, and induction of IgE-mediated immune responses determining prognosis, long-term outcome prediction or choosing (66). On the other hand, HDM requires uptake, processing and therapy (53, 54). When stratifying patients according to their sp IgE presentation by antigen presenting cells, a process that can be levels, the increased IL-31 in vitro response in patients with high favored by the presence of allergen-specific IgE on the surface of levels of sp IgE (over 100 kUA/L) supported the HDM contribution antigen presenting cells (67). Therefore, the IgE-mediated uptake to disease activity in AD (32). Noteworthy, the increased IL-31 and antigen presentation may be favored in group 1 of patients, mRNA expression in lesional skin from high sp IgE patients may since they present higher levels of HDM-specific IgE. explain why topical exposure to HDM induced IL-31 only in a subset Other inflammatory cells have been shown to produce IL-31: of HDM sensitized patients (21). The high sp IgE group also showed group 2 innate lymphoid cells (ILC2), basophils, eosinophils, enhanced plasma levels of periostin, CCL27, CCL18, CCL22 and sIL- dendritic cells, macrophages and mast cells (9–14) A single study 2R, thus associating the allergen-exposure and IL-31 response with has shown IL-31 expression by ILC2 (12), but the clinical relevance Th2-immune response and general inflammation (55). To evaluate of alarmins, which are needed for ILC2 activation, in AD is not clear signs of HDM clinical symptoms and to compare the reactivity of since all targeted therapies against them failed in clinical trials (68). CLA+ memory T cells to HDM with other allergens may be Regarding basophils, IL-31 has been shown in chronic spontaneous considered to complement these current findings. urticaria and healthy donors (10), and no study using AD-derived Frontiers in Immunology 09 frontiersin.org Sans-de San Nicolàs et al. 10.3389/fimmu.2023.1124018 basophils has shown IL-31 production. In eosinophils, IL-3 induces Santa Creu I Sant Pau (Spain). Participants provided written IL-31 production, but clinical relevance of eosinophils in AD has informed consent to participate in this study. not been confirmed by anti-IL-5 therapy (14, 68). Dendritic cells express a hundred times less IL-31 than Th2 cells and whether dendritic cells produce physiologically relevant IL-31 quantities is Author contributions underexplored (9). Recently, a new network comprising IL-31+ M2 macrophages, basophils, periostin and TSLP has been described LS-dSN, IF-N, RP and LS-B contributed to the design of the study. (13); nonetheless, the clinical relevance of these results are unknown IF-N, MB-O, AG, LC-B, MB-C, MF and ES-B included participants. due to lack of efficacy of TSLP blockade for AD treatment (68, 69). LS-dSN, IF-N and IG-J participated in the investigation. LS-dSN Mast cells from psoriatic skin and healthy donors express IL-31, but analyzed the data and prepared figures. LS-dSN and LS-B wrote the this expression has not been shown for AD (11). Altogether, manuscript. MF, RP and LS-B contributed to the funding acquisition. although IL-31 can be produced by different cell sources besides RP and LS-B supervised the study. All authors contributed to the article CLA+ memory T cells, no association with clinical pruritus has been and approved the submitted version. reported, therefore their translational relevance is still under investigation. Additional studies on the involvement of IL-31 production by these non-CLA+ memory T cells on epithelial cells Funding in coculture assays may be of help to further understand more mechanisms of IL-31 in AD. The study was funded by FIS/ISCIII 2021 (Ministerio de This study has some limitations. We tried to detect IL-31 by Economı́a y Competitividad e Instituto de Salud Carlos III; PI21/ intracellular flow cytometry but it was a complex issue due to the 01179 and PI21/00335) and Fondo Europeo de Desarrollo Regional lack of good tools (5), and plasma or serum IL-31 levels were (FEDER). Additionally, Sans-de San Nicolàs L was granted by a difficult to detect. Additionally, sample size for gene-array analysis PhD fellowship from the Agency for Management of University and as well as qRT-PCR was limited and more detailed investigations on Research Grants of the Generalitat de Catalunya (FI-SDUR 2020); the interactions between lesional epidermal cells and CLA+ memory Garcı́a-Jimé nez I was granted by a PhD fellowship from the T cells are needed. Universitat de Barcelona (PREDOCS-UB 2020). Although the role of allergens in AD is still controversial and at the moment the stratification of response to anti-IL-31RA depending on IgE status has not been performed, it can be Conflict of interest hypothesized that assessing specific IgE status to HDM in candidates for anti-IL-31RA therapy may eventually be helpful AG has received grants from AbbVie, Janssen, Lilly, Novartis for identifying patients more prone to good clinical response to and Sanofi, and lecture fees from AbbVie, Almirall, Leo Pharma and anti-IL-31RA and with a more favorable response in comparison to Sanofi. ES-B is a consultant for AbbVie, Almirall, Galderma, dupilumab or JAK inhibitors. Genentec, Leo Pharma, Lilly, Novartis, Pfizer, Pierre Fabre and In summary, our findings bring new data on the mechanisms of Sanofi. LS-B is a consultant for AbbVie, Janssen, Leo Pharma, allergen sensitization and IL-31 production by memory T cells in Novartis and Sanofi. moderate-to-severe AD patients. The biphasic IL-31 response to The remaining authors declare that the research was conducted HDM demonstrated with this translational ex vivo model may in the absence of any commercial or financial relationships that explain the partial IL-31 expression after HDM topical exposure could be construed as a potential conflict of interest. in HDM-sensitized patients (21) and the heterogeneous response to anti-IL-31-directed therapies. Publisher’s note All claims expressed in this article are solely those of the authors Data availability statement and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the The original contributions presented in the study are publicly reviewers. Any product that may be evaluated in this article, or available in the Gene Expression Omnibus repository, accession claim that may be made by its manufacturer, is not guaranteed or number GSE226073. endorsed by the publisher. Ethics statement Supplementary material The study involved human participants and was reviewed and The Supplementary Material for this article can be found approved by the Ethics Committee in Hospital de Bellvitge, online at: https://www.frontiersin.org/articles/10.3389/ Hospital del Granollers, Hospital del Mar and Hospital de la fimmu.2023.1124018/full#supplementary-material Frontiers in Immunology 10 frontiersin.org Sans-de San Nicolàs et al. 10.3389/fimmu.2023.1124018 References 1. Steinhoff M, Ahmad F, Pandey A, Datsi A, AlHammadi A, Al-Khawaga S, et al. thickening that lead to impaired skin-barrier function. PloS One (2016) 11(8): Neuroimmune communication regulating pruritus in atopic dermatitis. J Allergy Clin e0161877. doi: 10.1371/journal.pone.0161877 Immunol (2022) 149(6):1875–98. doi: 10.1016/j.jaci.2022.03.010 23. Bilsborough J, Leung DYM, Maurer M, Howell M, Boguniewcz M, Yao L, et al. 2. Datsi A, Steinhoff M, Ahmad F, Alam M, Buddenkotte J. 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