Possible Involvement of Antigen-Presenting Cells Expressing the Macrophage Galactose-Type C-Type Lectin in Inflammatory Skin Diseases

Atopic dermatitis (AD) and psoriasis (Pso) are common systemic inflammatory skin diseases in which dendritic cells (DCs) and macrophages (MØs) play central roles. Clarifying the cell subsets that are involved in the pathogenesis of AD and Pso is a prerequisite to developing effective treatments. Our research focuses on macrophage galactose-type C-type lectin (MGL)/CLEC10A/CD301 (referred to as MGL in the remaining parts of this paper), which is expressed on limited subpopulations of DCs and MØs but not on Langerhans cells (Kumamoto et al., 2009Kumamoto Y. Denda-Nagai K. Aida S. Higashi N. Irimura T. MGL2 Dermal dendritic cells are sufficient to initiate contact hypersensitivity in vivo.PLoS One. 2009; 4e5619Crossref PubMed Scopus (51) Google Scholar; Murakami et al., 2013Murakami R. Denda-Nagai K. Hashimoto S. Nagai S. Hattori M. Irimura T. A unique dermal dendritic cell subset that skews the immune response toward Th2.PLoS One. 2013; 8e73270Crossref Scopus (63) Google Scholar; Polak et al., 2014Polak M.E. Thirdborough S.M. Ung C.Y. Elliott T. Healy E. Freeman T.C. et al.Distinct molecular signature of human skin Langerhans cells denotes critical differences in cutaneous dendritic cell immune regulation.J Invest Dermatol. 2014; 134: 695-703Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) and is involved in the initiation of antigen presentation and signal transduction (Denda-Nagai et al., 2010Denda-Nagai K. Aida S. Saba K. Suzuki K. Moriyama S. Oo-Puthinan S. et al.Distribution and function of macrophage galactose-type C-type lectin 2 (MGL2/CD301b): efficient uptake and presentation of glycosylated antigens by dendritic cells.J Biol Chem. 2010; 285: 19193-19204Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar; Kanemaru et al., 2019Kanemaru K. Noguchi E. Tahara-Hanaoka S. Mizuno S. Tateno H. Denda-Nagai K. et al.Clec10a regulates mite-induced dermatitis [published correction appears in Sci Immunol 2020;5:eabg0688].Sci Immunol. 2019; 4eaax6908PubMed Google Scholar; Kurashina et al., 2021Kurashina R. Denda-Nagai K. Saba K. Hisai T. Hara H. Irimura T. Intestinal lamina propria macrophages upregulate interleukin-10 mRNA in response to signals from commensal bacteria recognized by MGL1/CD301a.Glycobiology. 2021; 31: 827-837Crossref PubMed Scopus (3) Google Scholar). Mouse MGL2/CD301b is likely to be the orthologous protein of human MGL. MGL2+ cells are involved in the skewing of the immune response toward T helper (Th) 2 in mice (Gao et al., 2013Gao Y. Nish S.A. Jiang R. Hou L. Licona-Limón P. Weinstein J.S. et al.Control of T helper 2 responses by transcription factor IRF4-dependent dendritic cells.Immunity. 2013; 39: 722-732Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar; Kumamoto et al., 2013Kumamoto Y. Linehan M. Weinstein J.S. Laidlaw B.J. Craft J.E. Iwasaki A. CD301b+ dermal dendritic cells drive T helper 2 cell-mediated immunity.Immunity. 2013; 39: 733-743Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar; Murakami et al., 2013Murakami R. Denda-Nagai K. Hashimoto S. Nagai S. Hattori M. Irimura T. A unique dermal dendritic cell subset that skews the immune response toward Th2.PLoS One. 2013; 8e73270Crossref Scopus (63) Google Scholar). In addition, MGL2+ DCs were significantly increased in psoriatic mouse skin and produced high levels of IL-23, which is associated with the pathogenesis of Pso (Kim et al., 2018Kim T.G. Kim S.H. Park J. Choi W. Sohn M. Na H.Y. et al.Skin-specific CD301b+ dermal dendritic cells drive IL-17-mediated psoriasis-like immune response in mice.J Invest Dermatol. 2018; 138: 844-853Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Such immune modulatory involvement of MGL+ cells is potentially conserved in humans. Recent reports have shown that MGL-positive human type 2 DCs (i.e., cDC2) express CD1c, CD14, and CD163 (Bourdely et al., 2020Bourdely P. Anselmi G. Vaivode K. Ramos R.N. Missolo-Koussou Y. Hidalgo S. et al.Transcriptional and functional analysis of CD1c+ human dendritic cells identifies a CD163+ subset priming CD8+CD103+ T cells.Immunity. 2020; 53: 335-352.e8Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar; Brown et al., 2019Brown C.C. Gudjonson H. Pritykin Y. Deep D. Lavallée V.P. Mendoza A. et al.Transcriptional basis of mouse and human dendritic cell heterogeneity.Cell. 2019; 179: 846-863.e24Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar; Heger et al., 2018Heger L. Balk S. Lühr J.J. Heidkamp G.F. Lehmann C.H.K. Hatscher L. et al.CLEC10A is a specific marker for human CD1c+ dendritic cells and enhances their toll-like receptor 7/8-induced cytokine secretion.Front Immunol. 2018; 9: 744Crossref PubMed Scopus (72) Google Scholar). In addition, it has been reported that MGL+CD14+cDC2s were increased in inflammation and were related to Th17 cell differentiation in Pso (Nakamizo et al., 2021Nakamizo S. Dutertre C.A. Khalilnezhad A. Zhang X.M. Lim S. Lum J. et al.Single-cell analysis of human skin identifies CD14+ type 3 dendritic cells co-producing IL1B and IL23A in psoriasis.J Exp Med. 2021; 218e20202345Crossref PubMed Scopus (48) Google Scholar). However, the distribution, phenotype, and function of MGL+ cells in human skin remain largely unknown. In this study, we used biopsy specimens to identify human MGL+ cells in healthy (healthy controls [HCs], n = 12), AD (n = 11), and Pso (n = 16) skin. Subject characteristics are shown in Supplementary Table S1. Materials and Methods can be found in the Supplementary information. MGL+ cells were observed in both normal and pathological skin, mainly in the upper dermis, especially around vessels (Supplementary Figure S1 and Figure 1a−c ). In both AD and Pso, MGL+ cells were observed in the epidermis and dermis (Figure 1b and c), whereas in HCs, MGL+ cells were rare in the epidermis (Figure 1a). No staining was observed with a control antibody (Supplementary Figure S2a). Quantification of cell numbers showed that MGL+ cell density was increased in AD and Pso compared with that in HCs (Figure 1d−f), suggesting that MGL+ cells are recruited or that MGL expression is induced during inflammation. Supportive analysis of two publicly available datasets—GSE121212 and E-MTAB-8149—that investigated AD, Pso, and HC skin biopsy specimens revealed that the MGL mRNA expression level was significantly higher in AD than in HC and in Pso than in HC specimens (Supplementary Results and Supplementary Figure S3). Next, we investigated the correlation of MGL+ cells with clinical parameters, including TARC/CCL17, a Th2 chemokine involved in allergic inflammation (Saeki and Tamaki, 2006Saeki H. Tamaki K. Thymus and activation regulated chemokine (TARC)/CCL17 and skin diseases.J Dermatol Sci. 2006; 43: 75-84Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). In AD skin, the number of MGL+ cells in the dermis positively correlated with serum TARC/CCL17 levels, serum IgE levels, and the number of TARC+ cells in the dermis (Figure 1g−i). In the AD epidermis, no significant correlation with these markers was observed (Figure 1k−m). TARC/CCL17 serum levels correlated with the number of TARC+ cells in the upper dermis but not in the epidermis (Supplementary Figure S4). There was no significant correlation between the Eczema Area and Severity Index and the number of MGL+ cells (Supplementary Figure S2b and c). MGL+ cells are not limited to a single-cell population, and patients with AD show some heterogeneity; for instance, some patients shift from a Th2 type to a Th1 type during the chronic cutaneous phase (Kataoka, 2014Kataoka Y. Thymus and activation-regulated chemokine as a clinical biomarker in atopic dermatitis.J Dermatol. 2014; 41: 221-229Crossref PubMed Scopus (107) Google Scholar). Therefore, MGL+ cell subsets should be investigated more systematically and in a larger number of subjects. Regarding Pso skin, the number of TARC+ cells correlated with the number of MGL+ cells in the epidermis and dermis (Figure 1j and n). The Psoriasis Area Severity Index did not correlate with the number of MGL+ cells (Supplementary Figure S2d and e). To characterize the subsets of MGL+ DCs and MØs, we used antibodies specific for MGL and CD1c, CD1a, CD14, and CD68. The major double-stained cell populations in HC, AD, and Pso dermis were CD1c+MGL+ cells (Figure 2a and b and Supplementary Figure S5a and b). In AD and Pso, a few CD1a+MGL+, CD14+MGL+, and CD68+MGL+ cells were also observed in the upper dermis (Figure 2a and b and Supplementary Figure S5a and b). The percentage of MGL+CD14+ cells in the dermis was significantly higher in AD and Pso than in HC (Supplementary Figure S6). Previously, CD1c+ DCs and CD14+ MØs were reported in the steady state, and monocyte-derived DCs increased during inflammation (Kashem et al., 2017Kashem S.W. Haniffa M. Kaplan D.H. Antigen-presenting cells in the skin.Annu Rev Immunol. 2017; 35: 469-499Crossref PubMed Scopus (210) Google Scholar). Our data suggest that MGL expression is not limited to a single-cell subset but that MGL+ cells are comprised of CD1c+ DCs and a few CD1a+ DCs and CD14+DCs or MØs in the dermis of human skin. In AD and Pso, CD1c+MGL+ cells were also observed in the epidermis, together with a few CD1a+MGL+ and CD14+MGL+ cells (Figure 2b, bottom panels, and Supplementary Figure S5b). This agrees with a previous report (Guttman-Yassky et al., 2007Guttman-Yassky E. Lowes M.A. Fuentes-Duculan J. Whynot J. Novitskaya I. Cardinale I. et al.Major differences in inflammatory dendritic cells and their products distinguish atopic dermatitis from psoriasis.J Allergy Clin Immunol. 2007; 119: 1210-1217Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Furthermore, in 9 of 11 AD specimens, a total of 14 spongiotic areas in the epidermis were found to be infiltrated by MGL+ cells, similar to those shown in Figure 1b. Recent reports show that inflammatory dendritic epidermal cells accumulate in and Langerhans cells surround spongiotic areas in skin lesions of patients with AD (Tanei and Hasegawa, 2022Tanei R. Hasegawa Y. Immunological pathomechanisms of spongiotic dermatitis in skin lesions of atopic dermatitis.Int J Mol Sci. 2022; 23: 6682Crossref PubMed Scopus (6) Google Scholar, Tanei and Hasegawa, 2021Tanei R. Hasegawa Y. Immunohistopathological analysis of immunoglobulin E-positive epidermal dendritic cells with house dust mite antigens in naturally occurring skin lesions of adult and elderly patients with atopic dermatitis.Dermatopathology (Basel). 2021; 8: 426-441Crossref PubMed Google Scholar). Therefore, it is plausible that MGL+ cells might be involved in the formation and/or exacerbation or the suppression of spongiotic lesions as Langerhans cell−like cells or inflammatory dendritic epidermal cells. Next, we stained HC, AD, and Pso patient skin sections with antibodies specific for MGL and TARC/CCL17 and counted the number of MGL+TARC+ double-positive cells. AD and Pso specimens contained significantly more MGL+ cells, which were costained with TARC/CCL17, than HC specimens (Figure 2c and Supplementary Figures S5c and S6). In particular, 65.7% of MGL+ cells in the AD dermis were costained with TARC/CCL17. In Pso, 42.3% of MGL+ cells in the epidermis and 28.6% of MGL+ cells in the dermis were costained with TARC/CCL17. The prevalence of MGL+ cells among TARC+ cells was 59.0% in the AD dermis and 66.7% in the Pso dermis. These findings suggest that a portion of MGL+ cells coexpress TARC/CCL17 in both AD and Pso. Although Pso is regarded as a Th17-dominant disease, recent reports have shown that TARC/CCL17 levels are increased in subsets of patients with Pso, such as patients with severe pruritus (Nattkemper et al., 2018Nattkemper L.A. Tey H.L. Valdes-Rodriguez R. Lee H. Mollanazar N.K. Albornoz C. et al.The genetics of chronic itch: gene expression in the skin of patients with atopic dermatitis and psoriasis with severe itch.J Invest Dermatol. 2018; 138: 1311-1317Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar; Purzycka-Bohdan et al., 2022Purzycka-Bohdan D. Nedoszytko B. Zabłotna M. Gleń J. Szczerkowska-Dobosz A. Nowicki R.J. Chemokine profile in psoriasis patients in correlation with disease severity and pruritus.Int J Mol Sci. 2022; 2313330Crossref Scopus (5) Google Scholar). In conclusion, MGL+ cells are present in healthy skin dermis but are sparse in the epidermis, and they significantly increase in AD and Pso lesions, where they are present in both the epidermis and dermis and infiltrate spongiotic areas in AD skin lesions. Both DCs and MØs are likely to express MGL, with CD1c+MGL+ DCs being the most prominent. In addition, a subset of MGL+ cells in AD and Pso coexpress TARC/CCL17. Taken together, these findings encourage further exploration of the potential involvement of MGL+ cells in inflammatory skin diseases, including the possibility that MGL could serve as a therapeutic target. The study was approved by St. Luke’s International Hospital Ethics Committee (approval number R-1502) and conducted in accordance with the Declaration of Helsinki Principles. Written informed consent for participation in the study and publication of the results was obtained from all patients included in the study. All data are contained within this article and its Supplementary information files. Yukari Manome-Zenke: http://orcid.org/0000-0002-5352-6511 Kaori Denda-Nagai: http://orcid.org/0000-0001-8325-3289 Miki Noji: http://orcid.org/0000-0002-9768-8969 Naoto Tsuneda: http://orcid.org/0000-0002-5758-3855 Katrin Beate Ishii-Schrade: http://orcid.org/0000-0002-9167-8831 Ryuichi Murakami: http://orcid.org/0000-0001-8257-585X Naoki Kanomata: http://orcid.org/0000-0002-0172-4524 Satoru Arai: http://orcid.org/0000-0003-3632-4392 Tatsuro Irimura: http://orcid.org/0000-0002-4709-3131 Shigaku Ikeda: http://orcid.org/0000-0002-7520-0707 The authors state no conflict of interest. We wish to thank Shinya Yamahira (Center for Medical Sciences of St. Luke's International University, Tokyo, Japan) for providing valuable advice on confocal image analysis. This work was supported by the Japan Society for the Promotion of Science (KAKENHI grant numbers JP19K23852 and JP20K07163) and the Japan Agency for Medical Research and Development (grant number JP19ae0101026). Conceptualization: YMZ, KDN, TI; Data Curation: YMZ, RM; Formal Analysis: YMZ; Funding Acquisition: YMZ, KDN, TI; Investigation: YMZ, MN, NT; Methodology: YMZ, KDN; Project Administration: KDN; Resources: YMZ, MN; Supervision: KDN, NK, SA, TI, SI; Visualization: YMZ, KBIS; Writing – Original Draft Preparation: YMZ; Writing – Review and Editing: YMZ, KDN, KBIS, TI The sponsors had no role in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the article for publication. In the GSE121212 dataset, macrophage galactose-type C-type lectin (MGL) mRNA expression was upregulated in atopic dermatitis (AD) compared with that in healthy controls (HCs) (log2 fold change [FC] = 2.29, P = 1.27 × 10−26, false discovery rate [FDR] = 1.64 × 10−24) (Supplementary Figure S3a) and in psoriasis (Pso) compared with that in HCs (log2 FC = 1.40, P = 3.21 × 10−12, FDR = 1.75 × 10−11) (Supplementary Figure S3b). In the E-MTAB-8149 dataset, MGL mRNA expression was upregulated in AD compared with that in HCs (log2 FC = 1.25, P = 4.12 × 10−51, FDR = 5.79 × 10−49) (Supplementary Figure S3c) and in Pso compared with that in HCs (log2 FC = 0.74, P = 3.95 × 10−34, FDR = 3.62 × 10−33) (Supplementary Figure S3d). We collected human skin tissue samples from patients diagnosed with Pso (n = 16), patients diagnosed with AD (n = 11), and HCs (n = 12) who visited the Department of Dermatology at St. Luke’s International Hospital (Tokyo, Japan) during the period from March 2018 to October 2021. One specimen was taken per patient from the location with the most severe skin inflammation in the patient. All patients were asked to pause their skin medications for 1 week before the skin biopsy. For HC specimens, we obtained the residual sample from patients who visited our department for the resection of a noninflammatory benign skin tumor. Pediatric patients and patients with generalized pustular Pso were excluded from this study. Skin biopsies were taken, and peripheral blood was collected within a few days of the skin tissue sampling for the determination of serum TARC/CCL17 and total IgE levels. As a clinical severity score, Eczema Area and Severity Index of 0−72 for AD and Psoriasis Area and Severity Index of 0−72 for Pso were evaluated by a dermatologist. We previously established an antihuman MGL mAb (MLD-16) (Sano et al., 2007Sano Y. Usami K. Izawa R. Denda-Nagai K. Higashi N. Kimura T. et al.Properties of blocking and non-blocking monoclonal antibodies specific for human macrophage galactose-type C-type lectin (MGL/ClecSF10A/CD301).J Biochem. 2007; 141: 127-136Crossref PubMed Scopus (8) Google Scholar). We tested the applicability of MLD-16 on formalin-fixed paraffin-embedded tissue sections in comparison with a rabbit polyclonal antibody specific for MGL/CLEC10A (Atlas, Stockholm, Sweden) (Supplementary Figure S1) and examined the immunohistochemical localizations of MGL+ cells using the polyclonal antibody. For TARC/CCL17, a rabbit polyclonal antibody (Abcam, Cambridge, United Kingdom) was used as the primary antibody. The Ventana Optiview Detection Kit (Roche Diagnostics, Basel, Switzerland) was used as the secondary reagent. For immunofluorescence, MLD-16 and antibodies specific for CD1a (polyclonal rabbit, Atlas), CD1c (clone M241, Absolute Antibody, Wilton, United Kingdom), CD14 (clone SC69-02, Novusbio, Centennial, CO), and CD68 (clone EPR20545, Abcam) were used. Goat anti-mouse IgG (H+L) antibody Alexa Flour 488 (Jackson Immuno Research, West Grove, PA) and goat anti-rabbit IgG (H+L) antibody Alexa Flour 594 (Thermo Fisher Scientific, Waltham, MA) were used as secondary reagents. Vectashield Vibrance Antifade Mounting Medium with DAPI (Funakoshi, Tokyo, Japan) was used for DAPI staining. Skin samples were fixed in formalin, embedded in paraffin, and sectioned to 3 μm. Fixed sections were stained with MLD-16 monoclonal or anti-MGL/CLEC10A polyclonal antibodies and anti-TARC/CCL17 antibodies using an automatic staining machine (Ventana Benchmark Ultra, Roche Diagnostics). The number of MGL+ cells and TARC+ cells in different areas (five fields per slide) were determined. MGL+ cells were quantified separately in the epidermis and dermis. The cell numbers per unit area (cells/mm2) were counted manually using light microscopy (biological microscope BX53, Olympus, Tokyo, Japan) and analyzed using Fiji imaging software (Schindelin et al., 2012Schindelin J. Arganda-Carreras I. Frise E. Kaynig V. Longair M. Pietzsch T. et al.Fiji: an open-source platform for biological-image analysis.Nat Methods. 2012; 9: 676-682Crossref PubMed Scopus (34191) Google Scholar). Skin samples were frozen in Tissue-Tek O.C.T. compound (Sakura Finetek, Tokyo, Japan), and 4 μm thick sections were cut. The sections were fixed with cold methanol for 30 seconds and blocked with 3% BSA (Sigma-Aldrich, St. Louis, MO) in PBS (Nacalai Tesque, Kyoto, Japan) for 60 minutes at room temperature. For costaining with immune cell markers, the sections were incubated with MLD-16 mAb and antibodies specific for CD1a, CD1c, CD14, CD68, or TARC/CCL17 diluted in PBS containing 0.1 M calcium chloride (Nacalai Tesque) and 0.5M magnesium chloride (Nacalai Tesque) overnight at 4 C. After washing with PBS, the sections were incubated with secondary antibodies (anti-mouse IgG antibody Alexa Flour 488 and anti-rabbit IgG antibody Alexa Flour 594) for 30 minutes at room temperature in the dark. After washing with PBS, the sections were mounted with Vectashield Vibrance Antifade Mounting Medium, which contains DAPI as a nuclear stain. Stained sections were imaged, and pictures were taken by confocal microscopy (DMI6000b, Leica Microsystems, Wetzlar, Germany). The images were imported into Fiji image analysis software, and an area containing about 100 DAPI-stained cells was defined as one counting area. For AD and Pso specimens, areas comprising a lesion were selected. The number of DAPI+MGL+ and DAPI+MGL+ immune cell marker−positive cells per unit area was counted manually in three different areas per patient specimen. Specimens from three individuals were examined for each of the HC, AD, and Pso groups. Serum levels of TARC/CCL17 and total IgE were measured as part of the routine clinical diagnostic laboratory tests conducted at St. Luke’s International Hospital. Because the sample size of this study was relatively small, we sought to provide supportive evidence for increased MGL expression in AD and Pso on the mRNA level using publicly available RNA-sequencing and microarray data. We scrutinized the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/gds/) and the EBI ArrayExpress (https://www.ebi.ac.uk/arrayexpress/) databases for studies investigating differentially expressed genes in patients with AD and Pso and healthy volunteers. Selection criteria were (i) skin biopsy samples (ii) a large sample size, (iii) a comparable severity of AD and Pso (as judged by Severity Scoring of Atopic Dermatitis, Eczema Area and Severity Index, and Psoriasis Area and Severity Index scores) as in this study, (iv) a comparable age range as in this study, and (v) the availability of mRNA expression data on MGL and TARC/CCL17. Two datasets—Gene Expression Omnibus GSE121212 (n = 142) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE121212) and EBI ArrayExpress E-MTAB-8149 (n = 402) (www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-8149/)—fulfilled these criteria. Next, we confirmed that genes that had been previously reported as commonly upregulated in AD and Pso (Ghosh et al., 2015Ghosh D. Ding L. Sivaprasad U. Geh E. Biagini Myers J. Bernstein J.A. et al.Multiple transcriptome data analysis reveals biologically relevant atopic dermatitis signature genes and pathways.PLoS One. 2015; 10e0144316Crossref Scopus (61) Google Scholar; Tian et al., 2012Tian S. Krueger J.G. Li K. Jabbari A. Brodmerkel C. Lowes M.A. et al.Meta-analysis derived (MAD) transcriptome of psoriasis defines the "core" pathogenesis of disease.PLoS One. 2012; 7e44274Crossref Scopus (145) Google Scholar), such as KYNU, PI3, and IL17A, were also upregulated in GSE121212 and E-MTAB-8149. On the basis of this, we judged that the GSE121212 and E-MTAB-8149 datasets were sufficiently suitable for our purpose of gaining supportive evidence from publicly available data. Read count data of GSE121212 were downloaded from Gene Expression Omnibus, and differentially expressed genes in AD compared with those in HC and in Pso compared with those in HC were analyzed using edgeR (version 3.28.1) (McCarthy et al., 2012McCarthy D.J. Chen Y. Smyth G.K. Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation.Nucleic Acids Res. 2012; 40: 4288-4297Crossref PubMed Scopus (3061) Google Scholar; Robinson et al., 2010Robinson M.D. McCarthy D.J. Smyth G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.Bioinformatics. 2010; 26: 139-140Crossref PubMed Scopus (23336) Google Scholar) at criteria in which the FDR was <0.05, and the absolute log2 FC of gene expression was >0.5. CEL files of E-MTAB-8149 were downloaded from EBI ArrayExpress and processed using oligo (version 1.58.0) (Carvalho and Irizarry, 2010Carvalho B.S. Irizarry R.A. A framework for oligonucleotide microarray preprocessing.Bioinformatics. 2010; 26: 2363-2367Crossref PubMed Scopus (1045) Google Scholar). Annotation was added using pd.hugene.2.1.st (version 3.14.1) (Carvalho, 2015Carvalho B. Platform Design Info for Affymetrix HuGene-2_1-st.https://bioconductor.org/packages/release/data/annotation/html/pd.hugene.2.1.st.htmlDate: 2015Date accessed: May 1, 2022Google Scholar). Differentially expressed genes in AD compared with those in HC and in Pso compared with those in HC were analyzed using limma (version 3.50.3) (Ritchie et al., 2015Ritchie M.E. Phipson B. Wu D. Hu Y. Law C.W. Shi W. et al.limma powers differential expression analyses for RNA-sequencing and microarray studies.Nucleic Acids Res. 2015; 43: e47Crossref PubMed Scopus (17959) Google Scholar) at criteria in which the FDR was <0.05, and the absolute log2 FC of gene expression was >0.5. R (version 4.1 or 3.6) was used for the analysis. The Kruskal−Wallis test was used to analyze differences between MGL+ cell numbers in HC, AD, and Pso specimens. Spearman’s rank correlation was used to analyze the correlation between the number of MGL+ cells in the skin and serum TARC/CCL17, serum total IgE, and TARC+ cells in the skin. A P < 0.05 was considered statistically significant. Except where stated otherwise, all statistical analyses were performed with the Statistical Package for Social Sciences software, version 18.0 (SPSS, Chicago, IL).Supplementary Figure S2Distribution and density of MGL+ cells in human skin. (a) Control antibody staining in HCs and in patients with AD and Pso. Bar = 200 μm. (b, c) Correlation between the density of MGL+ cells and EASI. (b) AD dermis; (c) AD epidermis. (d, e) Correlation between the density of MGL+ cells and PASI. (d) Pso dermis; (e) Pso epidermis. Spearman’s rank correlation r is shown. AD, atopic dermatitis; EASI, Eczema Area and Severity Index; HC, healthy control; MGL, macrophage galactose-type C-type lectin; PASI, Psoriasis Area and Severity Index; Pso, psoriasis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S3Volcano plots of differentially expressed genes in skin biopsy specimens from patients with AD and Pso compared with those from HCs. Two publicly available datasets were analyzed: (a) GEO GSE121212 (AD vs. HC) and (b) GEO GSE121212 (Pso vs. HC) and (c) ArrayExpress E-MTAB-8149 (AD vs. HC) and (d) ArrayExpress E-MTAB-8149 (Pso vs. HC). The position of MGL is indicated. Blue: downregulated genes. Red: upregulated genes. AD, atopic dermatitis; FC, fold change; FDR, false discovery rate; GEO, Gene Expression Omnibus; HC, healthy control; MGL, macrophage galactose-type C-type lectin; Pso, psoriasis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S4Correlation between serum TARC levels and the numbers of TARC+ cells in atopic dermatitis skin. Each dot represents one patient. (a) Epidermis. (b) Dermis. r denotes Spearman's rank correlation coefficient.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S5Phenotypes of MGL+ cells in HC and AD and Pso skin. (a) Phenotypes of MGL+ cells in HC and AD skin. This figure shows the single-channel images for the merged images shown in Figure 2a. Skin biopsy specimens from HCs and patients with AD were costained with a mAb specific for MGL and antibodies specific for the innate immune cell markers CD1c, CD1a, CD14, and CD68. Specimens from three patients each were investigated, and representative images are shown. Arrowheads indicate double-stained cells. A thin, dotted line indicates the border between the dermis and the epidermis. Bar = 50 μm. (b) Phenotypes of MGL+ cells in HCs and Pso skin. This figure shows the single-channel images for the merged images shown in Figure 2b. Skin biopsy specimens from HC and patients with Pso were costained with a mAb specific for MGL and antibodies specific for the innate immune cell markers CD1c, CD1a, CD14, and CD68. Specimens from three patients each were investigated, and representative images are shown. Arrowheads indicate double-stained cells. A thin, dotted line indicates the border between the dermis and the epidermis. Bar = 100 μm. (c) Phenotypes of MGL+ cells in HC, AD, and Pso skin. This figure shows the single-channel images for the merged images shown in Figure 2c. Biopsy specimens from HCs and patients with AD and Pso were costained with a mAb specific for MGL and a polyclonal antibody specific for TARC/CCL17 and investigated by immunofluorescence. Specimens from three patients each were investigated, and representative images are shown. Arrowheads indicate double-stained cells. A thin, dotted line indicates the border between the dermis and the epidermis. Bar = 50 μm. AD, atopic dermatitis; HC, healthy control; MGL, macrophage galactose-type C-type lectin; Pso, psoriasis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S5Phenotypes of MGL+ cells in HC and AD and Pso skin. (a) Phenotypes of MGL+ cells in HC and AD skin. This figure shows the single-channel images for the merged images shown in Figure 2a. Skin biopsy specimens from HCs and patients with AD were costained with a mAb specific for MGL and antibodies specific for the innate immune cell markers CD1c, CD1a, CD14, and CD68. Specimens from three patients each were investigated, and representative images are shown. Arrowheads indicate double-stained cells. A thin, dotted line indicates the border between the dermis and the epidermis. Bar = 50 μm. (b) Phenotypes of MGL+ cells in HCs and Pso skin. This figure shows the single-channel images for the merged images shown in Figure 2b. Skin biopsy specimens from HC and patients with Pso were costained with a mAb specific for MGL and antibodies specific for the innate immune cell markers CD1c, CD1a, CD14, and CD68. Specimens from three patients each were investigated, and representative images are shown. Arrowheads indicate double-stained cells. A thin, dotted line indicates the border between the dermis and the epidermis. Bar = 100 μm. (c) Phenotypes of MGL+ cells in HC, AD, and Pso skin. This figure shows the single-channel images for the merged images shown in Figure 2c. Biopsy specimens from HCs and patients with AD and Pso were costained with a mAb specific for MGL and a polyclonal antibody specific for TARC/CCL17 and investigated by immunofluorescence. Specimens from three patients each were investigated, and representative images are shown. Arrowheads indicate double-stained cells. A thin, dotted line indicates the border between the dermis and the epidermis. Bar = 50 μm. AD, atopic dermatitis; HC, healthy control; MGL, macrophage galactose-type C-type lectin; Pso, psoriasis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S5Phenotypes of MGL+ cells in HC and AD and Pso skin. (a) Phenotypes of MGL+ cells in HC and AD skin. This figure shows the single-channel images for the merged images shown in Figure 2a. Skin biopsy specimens from HCs and patients with AD were costained with a mAb specific for MGL and antibodies specific for the innate immune cell markers CD1c, CD1a, CD14, and CD68. Specimens from three patients each were investigated, and representative images are shown. Arrowheads indicate double-stained cells. A thin, dotted line indicates the border between the dermis and the epidermis. Bar = 50 μm. (b) Phenotypes of MGL+ cells in HCs and Pso skin. This figure shows the single-channel images for the merged images shown in Figure 2b. Skin biopsy specimens from HC and patients with Pso were costained with a mAb specific for MGL and antibodies specific for the innate immune cell markers CD1c, CD1a, CD14, and CD68. Specimens from three patients each were investigated, and representative images are shown. Arrowheads indicate double-stained cells. A thin, dotted line indicates the border between the dermis and the epidermis. Bar = 100 μm. (c) Phenotypes of MGL+ cells in HC, AD, and Pso skin. This figure shows the single-channel images for the merged images shown in Figure 2c. Biopsy specimens from HCs and patients with AD and Pso were costained with a mAb specific for MGL and a polyclonal antibody specific for TARC/CCL17 and investigated by immunofluorescence. Specimens from three patients each were investigated, and representative images are shown. Arrowheads indicate double-stained cells. A thin, dotted line indicates the border between the dermis and the epidermis. Bar = 50 μm. AD, atopic dermatitis; HC, healthy control; MGL, macrophage galactose-type C-type lectin; Pso, psoriasis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S6Number of costained cells for each immune cell marker. (a, b) Semiquantitative data from the double-staining immunohistochemistry experiment shown in Figure 2. Skin biopsy specimens from HCs and patients with AD and Pso were stained with antibodies specific to MGL, CD1a, CD1c, CD14, CD68, and TARC. The number of cells costained with anti-MGL antibody and each of the other immune cell markers was counted. The data represents the median (interquartile range = 25−75%) of three counting areas in one specimen from three patients each. Error bars show a 75% interquartile range. Analysis was performed with Kruskal−Wallis test: ∗P < 0.01 and ∗∗P < 0.001. AD, atopic dermatitis; HC, healthy control; MGL, macrophage galactose-type C-type lectin; Pso, psoriasis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Table S1Subject CharacteristicsNoAge/SexDiagnosisSerum Total IgE (IU/ml)Serum TARC (pg/ml)TARC+Cells in Epidermis (Cells/mm2)TARC+Cells in Dermis (Cells/mm2)MGL+Cells in Epidermis (Cells/mm2)MGL+Cells in Dermis (Cells/mm2)PASI or EASI140/MHC————0.035.1—233/FHC————14.558.1—359/FHC————3.432.5—433/MHC————0.024.9—569/MHC————1.925.4—631/FHC————0.024.8—771/FHC————0.026.2—860/MHC————0.027.2—928/FHC————0.026.4—1051/MHC————0.024.3—1162/FHC————2.840.9—1243/MHC————0.022.3—1351/FAD>25,00013,2000.0105.77.5338.731.21454/MAD>25,00011,00065.7131.9252.2241.245.11571/MAD2,8003,26013.072.7110.3222.415.61641/MAD3,30011,60069.171.8118.0176.647.81748/FAD45048.929.559.0168.42.41846/MAD8202,1601.065.642.2160.823.41944/MAD7703324.836.574.2129.514.42047/FAD6,9008489.548.437.7129.521.52185/MAD8205,7505.389.095.6125.922.42244/MAD1801,62045.028.831.4104.217.02336/MAD—3827.542.513.378.130.12452/FPso——2.437.752.0146.029.72531/MPso——055.325.989.111.42662/MPso——7.423.0112.3114.317.42757/MPso——4.2126.735.3114.912.62841/MPso——3.814.515.681.039.62941/MPso——2.520.933.252.919.53027/FPso——6.243.262.650.86.83177/MPso——13.3124.483.9112.511.53235/MPso——2.357.3105.3215.01.63330/FPso——0.07.91.446.413.83467/MPso——1.634.38.5110.613.63543/FPso——0.034.93.827.71.63662/FPso——7.574.953.4139.37.63758/FPso——2.875.717.9117.17.23874/FPso——3.488.524.7162.65.03947/FPso——9.757.826.2128.612.0Abbreviations: AD, atopic dermatitis; EASI, Eczema Area and Severity Index; F, female; HC, healthy control; M, male; PASI, Psoriasis Area and Severity Index; Pso, psoriasis.A total of 39 patients—AD (n = 11), Pso (n = 16), and HC (n = 12)—were included in the analysis. The male:female ratios were 2:1 in AD, 1:1 in Pso, and 1:1 in HC, and the median age in each group was 49.5 years (interquartile range = 39.5−62.0) in AD, 47.0 years (interquartile range = 44.0−52.5) in Pso, and 47.0 years (interquartile range = 33.0−60.5) in HC. The median EASI and PASI scores were 22.4 (interquartile range = 16.3−30.65) and 11.8 (interquartile range = 7.1−14.7), respectively. Open table in a new tab Abbreviations: AD, atopic dermatitis; EASI, Eczema Area and Severity Index; F, female; HC, healthy control; M, male; PASI, Psoriasis Area and Severity Index; Pso, psoriasis. A total of 39 patients—AD (n = 11), Pso (n = 16), and HC (n = 12)—were included in the analysis. The male:female ratios were 2:1 in AD, 1:1 in Pso, and 1:1 in HC, and the median age in each group was 49.5 years (interquartile range = 39.5−62.0) in AD, 47.0 years (interquartile range = 44.0−52.5) in Pso, and 47.0 years (interquartile range = 33.0−60.5) in HC. The median EASI and PASI scores were 22.4 (interquartile range = 16.3−30.65) and 11.8 (interquartile range = 7.1−14.7), respectively.