Meisel et al. Microbiome (2018) 6:20 DOI 10.1186/s40168-018-0404-9 RESEARCH Open Access Commensal microbiota modulate gene expression in the skin Jacquelyn S. Meisel1, Georgia Sfyroera1, Casey Bartow-McKenney1, Ciara Gimblet1, Julia Bugayev1, Joseph Horwinski1, Brian Kim2, Jonathan R. Brestoff3, Amanda S. Tyldsley1, Qi Zheng1, Brendan P. Hodkinson1, David Artis4 and Elizabeth A. Grice1* Abstract Background: The skin harbors complex communities of resident microorganisms, yet little is known of their physiological roles and the molecular mechanisms that mediate cutaneous host-microbe interactions. Here, we profiled skin transcriptomes of mice reared in the presence and absence of microbiota to elucidate the range of pathways and functions modulated in the skin by the microbiota. Results: A total of 2820 genes were differentially regulated in response to microbial colonization and were enriched in gene ontology (GO) terms related to the host-immune response and epidermal differentiation. Innate immune response genes and genes involved in cytokine activity were generally upregulated in response to microbiota and included genes encoding toll-like receptors, antimicrobial peptides, the complement cascade, and genes involved in IL-1 family cytokine signaling and homing of T cells. Our results also reveal a role for the microbiota in modulating epidermal differentiation and development, with differential expression of genes in the epidermal differentiation complex (EDC). Genes with correlated co-expression patterns were enriched in binding sites for the transcription factors Klf4, AP-1, and SP-1, all implicated as regulators of epidermal differentiation. Finally, we identified transcriptional signatures of microbial regulation common to both the skin and the gastrointestinal tract. Conclusions: With this foundational approach, we establish a critical resource for understanding the genome-wide implications of microbially mediated gene expression in the skin and emphasize prospective ways in which the microbiome contributes to skin health and disease. Keywords: RNA sequencing, Cutaneous transcriptome, Germ-free mice, Host-microbe interactions, Skin microbiome Background Abnormal host-microbe interactions are associated As a barrier to the external environment, the skin har- with cutaneous disorders like atopic dermatitis, acne, bors microbial communities that are both topographic- and psoriasis [8–14], but the exact mechanisms under- ally diverse and temporally complex [1–4]. These lying the microbial contributions to disease development microbes are postulated to have important functions in and progression are currently unknown. Identifying the skin health [2], including colonization resistance to block complete range of host functions and pathways evoked invasion of pathogenic bacteria and regulation of the cu- by the skin microbiota will improve our understanding taneous inflammatory and immune response [5–7]. The of disease pathogenesis and reveal new preventative and skin must sense, interpret, and respond to microbial therapeutic targets. signals from the environment, orchestrating responses The full extent of cutaneous functions regulated by the appropriate for the stimuli while maintaining barrier skin microbiota remains unknown and previous work has function and protecting itself from pathogenic infection. focused heavily on characterizing the response of specific pathways to microbial colonization. Recent work in mouse models demonstrates that the commensal microbiota, * Correspondence: [email protected] along with hair follicle morphogenesis, is responsible for 1Department of Dermatology, University of Pennsylvania Perelman School of Medicine, 421 Curie Blvd, 1015 BRB II/III, Philadelphia, PA 19104, USA recruitment of regulatory T cells during neonatal life [15]. Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Meisel et al. Microbiome (2018) 6:20 Page 2 of 15 Regulatory T cells additionally establish and mediate im- isolated from murine germ free (GF; n = 9) and specific mune tolerance to skin commensal bacteria during a pathogen free, conventionally raised (SPF; n = 7) skin was defined window of development [16]. Skin commensal sequenced on the Illumina HiSeq 2000 to obtain over 1.2 bacteria also promote interleukin 1 (IL-1) signaling and ef- billion paired-end reads (median of 60 million reads per fector T cell functions, suggesting a role for the microbiota sample, see Additional file 1: Table S1 for sample summar- in driving and/or mediating inflammatory skin disorders ies) of good quality (Additional file 2: Figure S1A).Reads [6]. Other work has highlighted the contributions of spe- were mapped to the mouse reference genome using the cific types of bacteria in inducing T cell responses via in- STAR aligner [27], in conjunction with AlignerBoost [28]. teractions with skin-resident dendritic cell subsets [17]. Of reads that aligned to the mouse reference genome, an Complement, an ancient and evolutionarily conserved average of 88% of reads per sample were assigned to a fea- arm of the innate immune system, may also be regulated ture (Additional file 2: Figure S1B), and the majority in the skin by colonization with the commensal micro- mapped to protein coding RNA (Additional file 2:Figure biota [18]. While these studies and others have established S1C) with sufficient coverage (Additional file 2:Figure roles for the microbiota in shaping cutaneous immunity, S1D). Gene counts were filtered and normalized in the broad spectrum of host functions that are elicited NOISeq [29, 30], yielding a total of 15,448 features for and/or mediated by the microbiota, as well as their under- analysis (Additional file 3: Dataset S1). ARSyNseq [31] lying molecular mechanisms, remains uncharacterized. was used to control for batch effects associated with dif- Here, we aimed to identify the molecular signals that ferent sequencing runs (Additional file 4:FigureS2). mediate the cutaneous host response to the resident skin Biological replicates of GF and SPF skin cluster together microbiota on a genome-wide scale, thereby elucidating as demonstrated by non-metric multidimensional scaling the full range of cutaneous responses evoked by the (Fig. 1b). A total of 2820 genes were differentially expressed microbiome. We used sterile, germ-free mice that have between GF and SPF skin (FDR-corrected p value < 0.1, never been exposed to microbiota and compared their Fig. 1c, Additional file 3: Dataset S1). Of these, 730 cutaneous transcriptome to that of mice conventionally genesweredifferentiallyexpressedbyatwofold raised in the presence of microbiota. We reasoned that, difference or greater between GF and SPF skin: 408 up- similar to previous work performed in the gastrointes- regulated and 322 downregulated in the absence of tinal tract [19–25], this experimental design would allow microbiota. us to identify genes and pathways in the skin under tran- Weighted gene correlation network analysis (WGCNA) scriptional modulation by the microbiome. [32], an unsupervised method for correlating patterns of Differentially expressed genes were enriched for those gene expression, created a scale-free network with 13 cu- related to immunity and epidermal differentiation and de- taneous gene modules (Fig. 1d,Additionalfiles5 and 6: velopment. Further analysis revealed an enrichment of Figure S3A-C, Dataset S2). Ninety percent of all genes microbially regulated genes in the epidermal differenti- were assigned to modules, with the majority belonging to ation complex, a syntenic cluster of genes regulated in a modules M1 and M2 (5613 and 5259 genes, respectively). tissue-specific manner with critical roles in epidermal bar- Genes in each module were significantly enriched in dif- rier formation [26]. Analysis of coordinately regulated ferent biological processes, including RNA processing and genes suggests that genes under the transcriptional con- metabolic processes (M1), the immune response (M2), trol of Klf-4, AP-1, and SP-1 are microbially regulated. transport (M3), and inflammatory response and keratino- Finally, we identify genes that are similarly regulated by cyte differentiation (M4) (Additional file 7: Table S2). the microbiota in both the skin and gastrointestinal tract, While colonization status clustered closely to and was cor- highlighting commonalities in the molecular signals that related with the M4 module (ρ =0.45, p =0.08),onlythe govern host-microbe interactions at both barrier sites. M1 (ρ = − 0.58, p =0.02)andM2(ρ =0.6,p = 0.01) mod- Collectively, this work provides a critical foundation and ules were significantly correlated with the presence of resource for understanding cutaneous gene regulation by microbiota (Additional file 5: Figure S3D). The majority of the microbiota, while establishing the molecular signals differentially expressed genes (DEG) downregulated in governing host responses to microbial