1280  |     LETTERS TO THE EDITOR 4. Hawro T, Ohanyan T, Schoepke N, et al. The Urticaria Activity Score— Validity, Reliability, and Responsiveness. J Allergy Clin Immunol In Pract. 2018;6(4):1185-1190. 5. Bernstein JA, Singh U, Rao MB, Berendts K, Zhang X, Mutasim D. Benralizumab for Chronic Spontaneous Urticaria. N Engl J Med. 2020;383(14):1389-1391. 6. Kaplan AP, Joseph K, Maykut RJ, Geba GP, Zeldin RK. Treatment of chronic autoimmune urticaria with omalizumab. J Allergy Clin Immunol. 2008;122(3):569-573. 7. Kolkhir P, Church MK, Altrichter S, et al. Eosinopenia, in Chronic Spontaneous Urticaria, Is Associated with High Disease Activity, Autoimmunity, and Poor Response to Treatment. J Allergy Clin Immunol Pract. 2020;8(1):318-325. SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section. DOI: 10.1111/all.14744 Interplay between skin microbiota and immunity in atopic individuals To the Editor, The prevalence of allergic diseases continues to increase worldwide along with industrialization and urbanization. Emerging evidence provides insights into the connection between the development of chronic inflammatory disorders, such as asthma and allergies, and the human microbiota through the immune- modulatory function of the commensal microbes.1,2 In addition to the well- established evi- dence on the role of the gut microbiota in human health and disease, other microbial communities, such as cutaneous microbiota, play an important role in establishing and tuning the host immune system toward a balanced response against commensal and pathogenic microorganisms.3 Additionally, the skin microbiota may promote immune toler- ance toward environmental allergens both locally and systemically.4 Interindividual differences and alterations in the skin microbiota composition and its associations with human health and disease are evident, but a greater clarification of the factors that modify the composition of microbiota is needed.1,2 Factors that have been in- vestigated for their potential interaction with the microbiota include lifestyle, use of medication, and the exposure to a changing environ- mental biodiversity.1,5 Here, we explore the composition of skin microbiota in 62 healthy and atopic asymptomatic young adults, and associate that with the characteristics of their childhood and present living en- vironments, allergen- specific serum IgE levels, and the immune function of their peripheral blood mononuclear cells (PBMCs) (see Figure 1A and supplementals for detailed methodological descrip- tion). We observed distinct associations between the skin micro- biota and allergen- specific serum IgE levels and the expression of cytokines, and identified several taxa that may play an important role in augmenting or suppressing the systemic inflammatory im- mune responses. Allergen- specific serum IgE measurements revealed that ap- proximately half of the subjects were sensitized to airborne and food allergens (aIgE > 0.35 kUA/L, n = 30), and of these, 43% were sensitized to birch pollen (bIgE > 0.35 kUA/L, n = 13), and 50% to timothy pollen (tIgE > 0.35 kUA/L, n = 15) (Figure S1A– C). Principal component analysis (PCA) of the allergen- specific IgE levels revealed clustering of all subjects, except those with very high (>12.5 kUA/L) aIgE, bIgE, or tIgE (Figure 1B), which are referred to as highly sensitized (HS; n = 11) individuals in sub- sequent analyses. Subjects with aIgE below 0.35 kUA/L are re- ferred to as non- sensitized (NS; n = 32) individuals. A total of 19 subjects exhibited intermediate allergen- specific IgE levels (0.35– 12.5 kUA/L). The relative levels of IL10, IL4, IL5, IL13, and IFNG mRNA were measured in PBMCs after 6 h of stimulation with either birch (Bet v 1) or timothy (Phl p 1) pollen allergen, and compared between the groups. HS individuals showed significantly higher expression of Th2- type cytokine IL4 in Bet v1 and Phl p 1 stimulated cells (p < 0.05) and Th1- type cytokine IFNG in Phl p 1 stimulated cells (p < 0.05) compared with NS. There was no difference in IFNG ex- pression between HS and NS after Bet v 1 stimulation. Furthermore, the expression of anti- inflammatory cytokine IL10 was significantly lower at the mRNA level in HS individuals compared with NS (in Phl p 1 stimulated PBMCs, p < 0.05; but not in Bet v 1 stimulated PBMCs, p < 0.1) (Figure 1C, results from Bet v 1 stimulated PBMCs in Figure S2).The mRNA levels correlated closely with protein levels measured in cell supernatants (Figure S3). In conclusion, we show that high allergen- specific serum IgE levels in our study subjects      |  1281LETTERS TO THE EDITOR are associated with potent pro- inflammatory, and attenuated anti- inflammatory responses to allergens. The skin microbiotas, analyzed by 16S rRNA gene sequenc- ing, mainly consisted of four phyla, including Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria (Figure 1D), repre- senting typical skin microbial communities consistent with previ- ous observations.2 We further explored the microbial compositions using distance- based redundancy analysis (dbRDA), testing the possible influence of living environments and allergen- specific IgE levels. Our results suggest that childhood living environments (urban, agricultural, or forest) may have impacted the skin micro- biota (Figure 2A) in a manner which is still discernible when con- straining the analysis by the effect of current living environment (Figure S4). Notably, the study subjects had moved from their first childhood homes since several years before the current study (Table S1). Finally, the constrained ordination analysis (dbRDA) suggested that high allergen- specific serum IgE levels may influence skin mi- crobial compositions (Figure 2B). However, the results did not reach statistical significance, likely due to the low number of HS individuals. To further explore associations between the skin microbiota and host immunity, we analyzed links between serum aIgE lev- els, cytokine expression in PBMCs, and the relative abundance of microorganisms, using Spearman's rank correlation. Our ex- ploratory analysis revealed several taxa, including Burkholderia– Caballeronia– Paraburkholderia, Ralstonia, Pelomonas, Curvibacter, Sediminibacterium, and Thermoactinomyces vulgaris, which correlated positively with aIgE levels and IL5 mRNA expression, and negatively with the expression ofIL10 (p < 0.05– 0.1) (Figure 2C– E, Table S2). Burkholderia– Caballeronia– Paraburkholderia, Ralstonia, Pelomonas, and Curvibacter are members of the Burkholderiaceae family, which includes organisms recognized as pathogens in the lungs, and an in- crease in their abundance has been associated with decreased pul- monary function in cystic fibrosis patients.5 Furthermore, T. vulgaris is known as one of the causative organisms in farmer's lung disease, which is a form of hypersensitivity pneumonitis.6 F I G U R E 1   Serum IgE levels, immune function, and skin microbiota composition. (A) Study population characteristics: group size (n), female ratio (F%), mean IgE levels specific for airborne and food (aIgE), birch (bIgE), and timothy (tIgE) allergens. (B) Principal component analysis of IgE levels (IgE < 12.5 kUA/L [blue]; >12.5 kUA/L [pink]). (C) IL10, IL4, and IFNG mRNA levels in Phlp1 stimulated peripheral blood mononuclear cells. P- values calculated with Wilcoxon rank- sum test. Log transformation using natural logarithm. (NS, non- sensitized; HS, highly sensitized individuals). (D) Taxa relative abundance at phylum level in non- sensitized (aIgE < 0.35 kUA/L) and sensitized (aIgE > 0.35 kUA/L) individuals † ≥ aIgE tIgE bIgE −2 0 2 0.0 2.5 5.0 7.5 PC1 (78.5%) PC 2 (21 .2% ) IgE (kUA/L) < 12.5 > 12.5 p = 0.044 5 6 7 8 NS HS lo g Re la tiv e Q ua nt ity IL−10 p = 0.026 NS HS IL−4 p = 0.024 NS HS IFNG Non−sensitized Sensitized 0.00 0.25 0.50 0.75 1.00 Ab u n da nc e Phylum Other Bacteroidetes Firmicutes Proteobacteria Actinobacteria †(A) (B) (D) (C) 1282  |     LETTERS TO THE EDITOR F I G U R E 2   Association between skin microbiota and immune function. (A, B) Distance- based redundancy analysis (dbRDA) constrained by (A) aIgE and childhood environment (aIgE, black lines; agriculture, yellow; forest, green; urban, purple; NS, non- sensitized, circles; HS, highly sensitized, triangles; intermediate IgE levels, squares), and by (B) IgE levels (aIgE, black lines; birch; timothy; HS, pink symbols, NS, light blue; intermediate, dark blue). (C) Hierarchical clustering of taxa correlating with aIgE and cytokines (rp; non- stimulated; ph, Phlp1; be, Betv1 stimulated peripheral blood mononuclear cells (*p < 0.05, **p < 0.01). (D– F) Spearman's rank correlation between the relative abundance of (D) Ralstonia and aIgE, (E) Pelomonas and IL10, (F) Anaerococcus and IL4 levels. Log transformation using natural logarithm −3 −2 −1 0 1 − 2 − 1 0 1 2 dbRDA1 db RD A2 IgE (kUA/L) > 12.5 (HS) 0.35 − 12.5 < 0.35 (NS) −2 −1 0 1 − 2 − 1 0 1 dbRDA1 db RD A2 Agriculture Forest Urban IgE (kUA/L) > 12.5 (HS) 0.35 − 12.5 < 0.35 (NS) rho = 0.53 P = 0.0045 −4 −2 0 2 4 1 2 Genus relative abundance sqrt(%) lo g aI gE (k UA /L ) Ralstonia rho = −0.44 P = 0.024 6 7 8 0.5 1.0 1.5 2.0 2.5 Genus relative abundance sqrt(%) lo g Re la tiv e Qu an tity IL 10 ph Pelomonas rho = −0.52 P = 0.0051 −2 0 2 4 0.0 0.1 0.2 0.3 Genus relative abundance sqrt(%) lo g Re la tiv e Q ua nt ity IL 4p h Anaerococcus a Ig E IL 5p h IL 4p h IF N G ph IL 10 ph IL 10 be IL 10 rp Anaerococcus Finegoldia Porphyromonas A lwoffii Veillonella Lactobacillus T vulgaris Sediminibacterium Pelomonas Ralstonia Curvibacter Burkholderia−C−P −0.6 −0.4 −0.2 0 0.2 0.4 0.6 Correlation coefficient * * * * * * * * * * ** * * ** ** * (A) (C) (D) (E) (F) (B)      |  1283LETTERS TO THE EDITOR In contrast, Acinetobacter lwoffii correlated positively with Phl p 1 induced IL10 (p < 0.01) and negatively with IL5 (p < 0.05), (Figure 2C, Table S2). Acinetobacter species are known to stimulate anti- inflammatory immune signaling in the skin, induce immune tolerance, and protect against allergies.4,7 Further, Porphyromonas, which negatively regulates airway allergic inflammation,8 correlated negatively with aIgE levels (p < 0.05).Moreover, Anaerococcus and Finegoldia correlated negatively with IL4 expression (Figure 2CF) (p < 0.01 and p < 0.05, respectively). These gram- positive anaerobic cocci have been found to be scarce in filaggrin- deficient ichthyosis vulgaris patients, suggesting that they may have protective effects in the skin.9 Furthermore, Lactobacillus and Veillonella correlated positively with IFNG and IL10 expression, respectively (p < 0.05). An increase in Veillonella abundance is associated with decreased inflammation in the lungs of cystic fibrosis patients.5 In conclusion, we have identified distinct associations between the composition of the skin microbiota and immune signaling by comparing atopic individuals with healthy individuals. Our results suggest that the skin of allergen- sensitized asymptomatic individ- uals may be colonized by less favorable, pro- inflammatory microbial species, and is characterized by a general scarcity of beneficial anti- inflammatory microbes. ACKNOWLEDG MENTS We would like to pay our gratitude to our highly valued collaborator, Professor Ilkka Hanski, who inspired us, and helped us initiate this study, but sadly passed away before we were ready to publish our results. KE Y WORDS 16S rRNA gene sequencing, allergen- specific IgE, amplicon sequence variant (ASV), host– microbe interactions, skin microbiota FUNDING INFORMATION This research was supported by Jane & Aatos Erkko Foundation. CONFLIC T OF INTERE S T All authors declare no conflicts of interest. Matilda Riskumäki1,2 Ioannis Tessas3 Noora Ottman1 Alina Suomalainen2 Paulina Werner1 Piia Karisola2 Antti Lauerma3 Lasse Ruokolainen4 Antti Karkman5,6 Lukas Wisgrill1,7 Hanna Sinkko1,2 Jenni Lehtimäki8 Harri Alenius1,2 Nanna Fyhrquist1,2 1Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden 2Department of Bacteriology and Immunology, Medicum, University of Helsinki, Helsinki, Finland 3Skin and Allergy Hospital, Helsinki University Hospital, Helsinki, Finland 4Department of Biosciences, University of Helsinki, Helsinki, Finland 5Department of Microbiology, University of Helsinki, Helsinki, Finland 6Helsinki Institute of Sustainability Science (HELSUS), University of Helsinki, Helsinki, Finland 7Division of Neonatology, Pediatric Intensive Care and Neuropediatrics, Comprehensive Center for Pediatrics, Medical University of Vienna, Vienna, Austria 8Finnish Environment Institute SYKE, Helsinki, Finland Correspondence Nanna Fyhrquist, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden & Department of Bacteriology and Immunology, Medicum, University of Helsinki, Helsinki, Finland. Email: nanna.fyhrquist@ki.se ORCID Matilda Riskumäki https://orcid.org/0000-0002-1465-9055 Paulina Werner https://orcid.org/0000-0002-8112-0252 Piia Karisola https://orcid.org/0000-0003-0635-2704 Lasse Ruokolainen https://orcid.org/0000-0003-0951-9100 Lukas Wisgrill https://orcid.org/0000-0001-9833-9499 Harri Alenius https://orcid.org/0000-0003-0106-8923 R E FE R E N C E S 1. Huang YJ, Marsland BJ, Bunyavanich S, et al. The microbiome in al- lergic disease: current understanding and future opportunities— 2017 PRACTALL document of the American Academy of Allergy, Asthma & Immunology and the European Academy of Allergy and Clinical Immunology. J Allergy Clin Immunol. 2017;139(4):1099- 1110. https:// doi.org/10.1016/j.jaci.2017.02.007 2. Grice EA, Segre JA. The skin microbiome. Nat Rev Microbiol. 2011;9(4):244- 253. https://doi.org/10.1038/nrmic ro2537 3. Naik S, Bouladoux N, Wilhelm C, et al. Compartmentalized control of skin immunity by resident commensals. Science. 2012;337(6098):1115- 1119. https://doi.org/10.1126/scien ce.1225152 4. Fyhrquist N, Ruokolainen L, Suomalainen A, et al. Acinetobacter spe- cies in the skin microbiota protect against allergic sensitization and inflammation. J Allergy Clin Immunol. 2014;134(6):1301. https://doi. org/10.1016/j.jaci.2014.07.059 5. Hanski I, von Hertzen L, Fyhrquist N, et al. Environmental bio- diversity, human microbiota, and allergy are interrelated. PNAS. 2012;109(21):8334- 8339. https://doi.org/10.1073/pnas.12056 24109 6. Rossi GA, Morelli P, Galietta LJ, Colin AA. Airway microenvironment alterations and pathogen growth in cystic fibrosis. Pediatr Pulmonol. 2019;54(4):497- 506. https://doi.org/10.1002/ppul.24246 7. Erkinjuntti- Pekkanen R, Kokkarinen JI, Tukiainen HO, Reiman M, Terho EO. IgG antibodies, chronic 1284  |     LETTERS TO THE EDITOR bronchitis, and pulmonary function values in farmer’s lung patients and matched controls. Allergy. 1999;54(11):1181- 1187. https://doi. org/10.1034/j.1398- 9995.1999.00275.x 8. Card JW, Carey MA, Voltz JW, et al. Modulation of allergic airway inflammation by the oral pathogen Porphyromonas gingivalis. Infect Immun. 2010;78(6):2488- 2496. https://doi.org/10.1128/IAI.01270 - 09 9. Zeeuwen PLJM, Ederveen THA, van der Krieken DA, et al. Gram- positive anaerobe cocci are underrepresented in the microbiome of filaggrin- deficient human skin. J Allergy Clin Immunol. 2017;139(4):1368- 1371. https://doi.org/10.1016/j.jaci.2016.09.017 SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section.