Microbiome dynamics of human epidermis following skin barrier disruption

Patrick L J M Zeeuwen, Jos Boekhorst, Ellen H van den Bogaard, Heleen D de Koning, Peter M C van de Kerkhof, Delphine M Saulnier, Iris I van Swam, Sacha A F T van Hijum, Michiel Kleerebezem, Joost Schalkwijk, Harro M Timmerman, Patrick L J M Zeeuwen, Jos Boekhorst, Ellen H van den Bogaard, Heleen D de Koning, Peter M C van de Kerkhof, Delphine M Saulnier, Iris I van Swam, Sacha A F T van Hijum, Michiel Kleerebezem, Joost Schalkwijk, Harro M Timmerman

Abstract

Background: Recent advances in sequencing technologies have enabled metagenomic analyses of many human body sites. Several studies have catalogued the composition of bacterial communities of the surface of human skin, mostly under static conditions in healthy volunteers. Skin injury will disturb the cutaneous homeostasis of the host tissue and its commensal microbiota, but the dynamics of this process have not been studied before. Here we analyzed the microbiota of the surface layer and the deeper layers of the stratum corneum of normal skin, and we investigated the dynamics of recolonization of skin microbiota following skin barrier disruption by tape stripping as a model of superficial injury.

Results: We observed gender differences in microbiota composition and showed that bacteria are not uniformly distributed in the stratum corneum. Phylogenetic distance analysis was employed to follow microbiota development during recolonization of injured skin. Surprisingly, the developing neo-microbiome at day 14 was more similar to that of the deeper stratum corneum layers than to the initial surface microbiome. In addition, we also observed variation in the host response towards superficial injury as assessed by the induction of antimicrobial protein expression in epidermal keratinocytes.

Conclusions: We suggest that the microbiome of the deeper layers, rather than that of the superficial skin layer, may be regarded as the host indigenous microbiome. Characterization of the skin microbiome under dynamic conditions, and the ensuing response of the microbial community and host tissue, will shed further light on the complex interaction between resident bacteria and epidermis.

Figures

Figure 1
Figure 1
Clustering, microbial community composition and microbial diversity of samples from different sampling sites. Composition is displayed as relative abundance, that is, the number of reads assigned to a genus divided by the total number of reads assigned up to the genus level. (a) Clustering of 20 samples of five healthy subjects. Samples were clustered using UPGMA with weighted UniFrac as a distance measure. The figure was generated with the interactive Tree of Life tool (iTOL) [70]. Participating volunteers are numbered HV1 to HV5 followed by the sampled body location. Colored bars represent the relative abundance of bacterial genera as determined by barcoded pyrosequencing (details in Materials and methods). (b) Phylogenetic diversity rarefaction curves for communities sampled from the listed skin locations show differences between armpit, forehead, inner elbow and upper buttock skin.
Figure 2
Figure 2
Redundancy analysis of the microbiota composition of the lower back of 12 adults for determining the most important variables (17 in total) explaining the variation in microbiota composition at the genus level. Genera that represented at least 85.1% of the first two principal components used as explanatory axis in the plots are shown as vectors. The first component and second component explain 32.6% and 24.4% of the variance, respectively. This figure was generated with Canoco version 4.5. The different variables are represented by arrows, where length reflects significance. Colors indicate sample groupings: red arrows represent all individuals, green arrows represent the males and females, and the blue arrows correspond to the stripping depth (STR0 is non-damaged healthy superficial skin, and STR5 and STR10 represent 5 and 10 times tape-stripped skin, respectively). The microbial genera are shown in black text color.
Figure 3
Figure 3
Clustering and microbial community composition of different volunteers and epidermal layers. Samples were clustered using UPGMA with weighted UniFrac as a distance measure. The figure was generated with iTOL [70]. Sample names with the same color come from the same volunteer (M = male 1 to 6, F = female 1 to 6), followed by the stripping depth (STR0, STR5, STR10). Colored bars represent the relative abundance (the number of reads assigned to a genus divided by the total number of reads assigned up to the phylum level) of bacterial genera as determined by barcoded pyrosequencing.
Figure 4
Figure 4
Difference in microbial community composition between males and females. Nodes represent taxa, edges link the different taxonomic levels. The fold increase is calculated as the 2log of the ratio of the relative in males and females (0 = no difference between genders, 1 = twice as abundant in female, and so on). The significance is expressed as the P-value of a Mann-Whitney U test of the male and female samples. Note that the relation between node-size and total abundance is non-linear.
Figure 5
Figure 5
Differences in microbial community composition between STR0 and STR10 samples. Nodes represent taxa, edges link the different taxonomic levels. The fold increase is calculated as the 2log of the ratio of the relative in males and females (0 = no difference between STR0 and STR10, 1 = twice as abundant in STR10, and so on). The significance is expressed as the P-value of a Mann-Whitney U test of the male and female samples. Note that the relation between node size and total abundance is non-linear.
Figure 6
Figure 6
Recolonization after stripping. The tree-like structure in the left side of the figure is the consensus tree. It was generated using consense (Phylip_REF) of the per-volunteer UPGMA UniFrac trees of the samples. The pie charts show the microbial community composition of the individual samples. Composition is displayed as relative abundance, that is, the number of reads assigned to a genus divided by the total number of reads assigned up to the genus level.
Figure 7
Figure 7
Variation in the antimicrobial protein host response upon superficial skin injury. (a) Variation in epidermal mRNA expression levels of eight genes that encode antimicrobial proteins, in normal healthy skin (NS) and upon tape stripping (TS) after 24 hours (n = 5). (b) hBD-2 and elafin protein expression levels before and after tape stripping in several individuals. Scale bar = 100 μm.
Figure 8
Figure 8
Model for skin injury, microbial recolonization, and host response. Details in Discussion and Conclusion sections. Recolonization is done by bacteria from the deeper layers of the adjacent skin (black arrows). During this process the microbial communities of the host and invading bacteria from the environment trigger the skin to express antimicrobial proteins and inflammatory molecules (yellow arrows). SC = stratum corneum; SG = stratum granulosum; SS = stratum spinosum.

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