Gram-positive anaerobe cocci are underrepresented in the microbiome of filaggrin-deficient human skin
Authors: Patrick L.J.M. Zeeuwen, Thomas H.A. Ederveen, Danique A. van der Krieken, Hanna Niehues, Jos Boekhorst, Sanja Kezic, Daniëlle A.T. Hanssen, Marisol E. Otero, Ivonne M.J.J. van Vlijmen-Willems, Diana Rodijk-Olthuis, Denise Falcone, Ellen H.J. van den Bogaard, Marijke Kamsteeg, Heleen D. de Koning, Manon E.J. Zeeuwen-Franssen, Maurice A.M. van Steensel, Michiel Kleerebezem, Harro M. Timmerman, Sacha A.F.T. van Hijum, Joost Schalkwijk
Journal: Journal of Allergy and Clinical Immunology (2016)

Next-generation sequencing technologies and powerful bioinformatics tools have revealed that diversity and composition of the skin microbiota of healthy volunteers strongly depends on the topographical location on the body and has a high degree of interpersonal variation.1Grice E.A. Segre J.A. The skin microbiome.Nat Rev Microbiol. 2011; 9: 244-253Crossref PubMed Scopus (1855) Google Scholar Nevertheless, the dominant types of bacteria remain relatively stable over time, and specific bacteria are associated with dry, moist, and/or sebaceous microenvironments.2Costello E.K. Lauber C.L. Hamady M. Fierer N. Gordon J.I. Knight R. Bacterial community variation in human body habitats across space and time.Science. 2009; 326: 1694-1697Crossref PubMed Scopus (2189) Google Scholar Microbial communities, genetic host factors, and the environmental factors at a particular moment constitute a complex relationship that is essential for skin barrier homeostasis.3Blekhman R. Goodrich J.K. Huang K. Sun Q. Bukowski R. Bell J.T. et al.Host genetic variation impacts microbiome composition across human body sites.Genome Biol. 2015; 16: 191Crossref PubMed Scopus (429) Google Scholar More recent studies have also focused on the microbiota of diseased and injured skin as alterations to microbial communities have been associated with cutaneous disorders.4Zeeuwen P.L. Kleerebezem M. Timmerman H.M. Schalkwijk J. Microbiome and skin diseases.Curr Opin Allergy Clin Immunol. 2013; 13: 514-520Crossref PubMed Scopus (112) Google Scholar, 5Zeeuwen P.L. Boekhorst J. van den Bogaard E.H. de Koning H.D. van de Kerkhof P.M. Saulnier D.M. et al.Microbiome dynamics of human epidermis following skin barrier disruption.Genome Biol. 2012; 13: R101Crossref PubMed Scopus (166) Google Scholar In the present study, we selected ichthyosis vulgaris (IV) as a model disease to investigate whether genetic polymorphisms resulting in altered stratum corneum (SC) composition and structure affect the microbiota composition of human skin and the cutaneous host response. Genetic deficiency or haploinsufficiency of the histidine-rich epidermal protein filaggrin (FLG) is associated with IV and atopic dermatitis (AD).6Smith F.J. Irvine A.D. Terron-Kwiatkowski A. Sandilands A. Campbell L.E. Zhao Y. et al.Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris.Nat Genet. 2006; 38: 337-342Crossref PubMed Scopus (812) Google Scholar, 7Palmer C.N. Irvine A.D. Terron-Kwiatkowski A. Zhao Y. Liao H. Lee S.P. et al.Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.Nat Genet. 2006; 38: 441-446Crossref PubMed Scopus (2281) Google Scholar Here, we report 2 novel findings with respect to FLG deficiency and cutaneous microbiota. First, we show that FLG deficiency is associated with a low relative abundance of proteolytic Gram-positive anaerobic cocci (GPAC), and a general underrepresentation of bacterial taxa that are capable of using histidine. Second, we report that exposure of cultured epidermal keratinocytes to Finegoldia magna induces the expression of antimicrobial proteins and proinflammatory cytokines, and that this response is distinct from other skin commensals such as Propionibacterium acnes, Corynebacterium aurimucosum, and Staphylococcus capitis, and the skin pathogen Staphylococcus aureus. All experimental procedures were performed as described in this article's Online Repository at www.jacionline.org. We analyzed the skin microbiota of the lower leg, which is typically a location where the ichthyotic skin alterations (dry and scaly skin) are most prominent. Patients and healthy controls were genotyped for FLG mutations (see Table E1 and Fig E1 in this article's Online Repository at www.jacionline.org). None of the patients with IV in our study had eczematous lesions present at the lower leg, thereby excluding (lesional) AD-associated microbiome alterations as a confounding factor. Biophysical measurements showed increased transepidermal water loss and decreased SC hydration in FLG−/− subjects compared with FLG+/+ subjects (see Fig E2 in this article's Online Repository at www.jacionline.org). Microbiome samples were subsequently analyzed by barcoded 16S marker gene sequencing (see Tables E2 and E3 in this article's Online Repository at www.jacionline.org). Rarefaction curves show that the phylogenetic diversity does not differ between FLG+/+, FLG+/−, and FLG−/− samples (see Figs E3 and E4 in this article's Online Repository at www.jacionline.org). Redundancy analysis revealed a significant effect of FLG deficiency on microbiota composition (see Fig E5 in this article's Online Repository at www.jacionline.org). A lower relative abundance of GPAC (average 1.7% of the total genera) was found in FLG−/− skin compared with FLG+/+ skin (average 9.3% of the total genera) (see Fig E6 and Excel file E1 in this article's Online Repository at www.jacionline.org). These genera included Finegoldia, Anaerococcus, and Peptoniphilus. Other highly abundant genera did not show significant differences between the genotypes (Excel file E1). We therefore focused on members of the GPAC group for further in silico analysis and experimental studies. Most GPAC are asaccharolytic and use the products of protein degradation as substrates for metabolic energy generation.8Murphy E.C. Frick I.M. Gram-positive anaerobic cocci–commensals and opportunistic pathogens.FEMS Microbiol Rev. 2013; 37: 520-553Crossref PubMed Scopus (192) Google Scholar The average relative quantities of these candidate-discriminating genera supported a significant difference between the FLG-deficient and FLG-proficient state, or more specifically, a strong decrease by FLG protein loss (Fig 1, A). Indeed, an in vitro survival disadvantage of F magna on FLG-deficient SC supported the in vivo findings (see Figs E7 and E8 in this article's Online Repository at www.jacionline.org). On the basis of bacterial 16S profiles of the skin microbiota, we tested the potential involvement of microbial reactions using natural moisturizing factors such as histidine, urocanic acid, and pyrrolidone carboxylic acid. Histidine is a known carbon source for bacteria that possess the histidine utilization (Hut) pathway (see Fig E9 in this article's Online Repository at www.jacionline.org), which is highly conserved among bacteria and found with high frequency in most phylogenetic groups within the bacterial domain. Contrast analysis of IV versus healthy skin with selected hut gene candidates (see Table E4 in this article's Online Repository at www.jacionline.org) revealed that hutU, hutI, and hutH orthologous genes were predicted to be significantly underrepresented in the microbiota of patients with IV (Fig 1, B; and Excel file E2 in this article's Online Repository at www.jacionline.org). In conclusion, our study reveals that FLG-deficient skin harbors significantly fewer bacterial taxa capable of using histidine as nutrient (carbon) source, which we postulate to be a consequence of nutrient competition driven by the loss of the histidine-rich protein FLG. Because microbial factors such as S aureus colonization have been implicated in AD,9Kong H.H. Oh J. Deming C. Conlan S. Grice E.A. Beatson M.A. et al.Temporal shifts in the skin microbiome associated with disease flares and treatment in children with atopic dermatitis.Genome Res. 2012; 22: 850-859Crossref PubMed Scopus (1105) Google Scholar we explored possible interactions of this pathogen and several skin commensals, including F magna, with epidermal keratinocytes (Fig 2; and Table E5 in this article's Online Repository at www.jacionline.org). F magna induced a significantly higher expression of antimicrobial peptides (AMPs) compared with S aureus and the other skin commensals. For S aureus and S capitis, keratinocyte stimulation caused a significantly higher expression of proinflammatory cytokines than was observed for stimulation with F magna. Our observation that keratinocytes appear to exhibit different cytokine/AMP responses toward S aureus or F magna stimulation is intriguing. Given the known functions and activities of IL-8 (neutrophil chemoattractant), IL-6 (inducer of inflammatory responses), and the chemokine ligand 20 (CCL20, chemoattractant for various white blood cell types, antimicrobial activity), their strong induction by S aureus contributes to control of infection. The observed rapid induction of AMPs by F magna, however, suggests that this may be an important signaling mechanism to the keratinocytes when the skin barrier is breached and a commensal bacterium comes into close contact with the epidermal keratinocytes. Complete or partial absence of F magna could cause an impaired or delayed danger signaling to the keratinocytes in FLG−/− or FLG+/− individuals. This could, speculatively, be a mechanism that favors S aureus colonization or infection, but clearly requires further investigation. In conclusion, we have uncovered new and potentially important biological aspects of a very common genetic polymorphism. Our study has shown this to be the case for the homozygous-deficient FLG state, but our data on heterozygotes suggest a gene dosage effect. This generates new and testable hypotheses to investigate a contribution of microbiome alterations in the pathogenesis of AD. We thank all patients and volunteers who participated in this study. Download .docx (5.33 MB) Help with docx files Online Repository text Download .xlsx (.18 MB) Help with xlsx files Excel E1 Download .xlsx (5.01 MB) Help with xlsx files Excel E2