How do the skin barrier and microbiome adapt to the extra‐uterine environment after birth? Implications for the clinical practice

The multiple protective functions of the skin derive from the interactions between epithelial skin and immune cells as well as the commensal microbiota. Developed in the last trimester of intra‐uterine life, the skin barrier adapts dynamically after birth. Specific differences in the structure and physiology have been disclosed between infant and adult skin. The stratum corneum of infants is thinner and structured by thicker corneocytes with a more anisotropic surface in comparison to adult skin. Lower levels of the natural moisturizing factor and its constituents, together with the increased protease activity in the epidermis result in dry baby skin and ongoing adaptation of the desquamation to the extra‐uterine environment. Infant epidermis is characterized by an accelerated proliferation rate and clinically competent permeability barrier in term neonates, despite the higher baseline values of transepidermal water loss in infants. The skin surface of newborns is less acidic, which could increase susceptibility to diaper and atopic dermatitis. Immediately after birth, skin is colonized by commensal bacteria—a process dependent on the mode of delivery and of major importance for the maturation of the immune system. Skin bacterial diversity and dysbiosis have been related to different pathology such as atopic and seborrheic dermatitis. This paper focuses on the ongoing structural, functional and biochemical adaptation of the human skin barrier after birth. We discuss the interactions on the ‘skin barrier/ microbiota/ immune system’ axis and their role in the development of competent functional integrity of the epidermal barrier.


INTRODUCTION
Human skin with its outermost layers accomplishes a variety of functions, for example the permeability barrier and multiple defence mechanisms against external stressors. After birth, the skin of term newborn children represents a competent barrier to outside-in exogenous factors and an inside-out barrier to ensure homeostatic functions [1,2]. Several studies have shown that the adaptation of the skin barrier to the dry extra-uterine surrounding environment is yet not accomplished after birth [3][4][5][6]. The first consecutive months are critical for the adaptation from aqueous intra-uterine life to an oxygen-rich environment.
Non-invasive methods have been employed in studying the process of adaptation after birth such as the assessment of transepidermal water loss (TEWL) and skin surface, capacitance-based corneometry, in vivo Raman spectroscopy and scanning electron microscopy. By using these methods, a growing body of evidence suggests that the skin barrier is a subject of ongoing adaptation concerning its structure and functions after birth. Infant stratum corneum (SC) structure shows differences in dermal papillae density and distribution compared to those of adults [5]. A possible consequence of these discrepancies on the functional level is the not fully competent water-handling properties despite the intact SC of newborns [7][8][9].
We perceive the skin surface microbiome as a substantial part of the skin barrier. Emerging reports suggest that a shift in skin microbiome diversity could predispose to certain diseases such as atopic dermatitis (AD) [10]. Being relatively uniform at birth, the skin microbiome is adapting in the period of infancy and is related to the mode of delivery [11].

STRUCTURAL ADAPTATION AFTER BIRTH
Infant epidermis shows specific differences on the structural level in comparison to adults [12,13]: the SC of young children is 30% thinner than adult SC [14]. Morphologically, surface glyphs are denser packed in young children compared to adult skin, and the corresponding interglyphal
In preterm neonates, the papillary dermis is oedematous and the collagen fibres and anchoring filaments are smaller than those in adults or term neonates [16]. The capillary network is not yet organized after birth and the microvasculature shows a horizontal plexus and capillary network [17].
Quantitative analysis of the morphological SC surface pictures (volar forearm) obtained with scanning electron microscopy (SEM) was investigated regarding the postnatal evolution patterns of the skin surface [18]. A semi-quantitative score for surface evaluation (electron microscopy isotropy; EMI) was generated, assessing the pattern of SC micro-morphology organization. Lower EMI values represent a less mature SC surface morphology. Newborn children presented with the lowest EMI values of approx. three points. The EMI score increased with age. Children in the age groups 6 months, 1 year as well as 4-5 years had lower EMI scores than adults. Correlation calculations showed a link between EMI score values and age. Children younger than 2 demonstrated a rapid increase of EMI values of 4 EMI points compared to younger children of 1-15 days. The comparison of 2 years old children to adults showed a slower EMI score increase rate of 2 EMI points. This data is indicative of a maturation process of the skin surface micromorphology that reaches a plateau around the age of 2 years.
Quantitative studies looked at the distribution of corneodesmosome remnants [18]. In the group of 5-6 weeks infants, corneodesmosin labelling on the outside of corneocyte clusters was diminished. No trace of the 'central' corneodesmosomal labelling was detectable in newborns' surface corneocytes. The distribution pattern in irregularly dispersed corneoycte agglomerates is indicative of incomplete keratinization and desquamation process. In these areas, corneodesmosome degradation takes place only in isolated areas and is incomplete.

BIOCHEMICAL COMPOSITION ADAPTATION
Skin barrier biochemical composition is also adapting to the extra-uterine environment after birth. Elevated protease activity, namely chymotrypsin-like protease activity, and lower NMF levels were observed parallel to perturbed barrier function in neonates and at the age of one month [19]. The authors concluded that the processes of keratinization and desquamation are not fully developed in infant skin. Others could not determine any substantial difference in chymotrypsin-like activity between infants and adults in an East Asian cohort [20].
In infants, proteins involved in late differentiation, cornification and filaggrin processing, for example serine proteases and anti-microbial peptides (S100 and MPO), were increased in comparison to adults [21].
In vivo Raman confocal microscopy (RCM) allows the profiling of epidermal molecular components [22,23]. The concentration profiles of epidermal ceramides, sweat constituents (e.g. urea and lactate), natural moisturizing factors and specific water profiles are assessed noninvasively. Furthermore, the modulation of the profiles was studied under the impact of exogenous conditions [7,9,22,[24][25][26]. Infants showed a sharper increase in the water gradient and higher water content in the epidermis compared to adults [7]. A lower NMF and ceramide content was observed in young children compared to adult epidermis [7,27,28]. In addition, ethnic variations in SC ceramide levels have been described [29].
A similar increase in the water content related to skin depth was detectable for infants and adults. However, the depth-dependent water content increase was lower in newborns [30]. Newborns showed the highest mean AUCs values for NMF in Raman profiles, reaching a plateau at the age of 6 months. The analysis of the separate NMF constituents (pyrrolidone carboxylic acid, serine, glycine, histidine, urea and trans-uranic acid) showed similar profiles compared to bulk NMF [30]. The mean AUC values for lactic acid were not increased in newborns compared to older children. Only slight differences between the different age groups regarding the mean AUCs of Raman depth profiles for ornithine, proline and alanine were observed [30]. The increased NMF is a compensatory mechanism for the decreased SC hydration at birth. Lower NMF in infants with certain regional variations in the maturation of the histidine/urocanic acid ratio and the trans-urocanic acid/cis-urocanic acid ratio together with an increase in plasmin activity with age have been witnessed [15,31].

Permeability barrier evaluation by TEWL measurement
TEWL as a surrogate marker for epidermal barrier maturation is related to gestational age (GA) and postnatal age (PNA) [32][33][34]. The concept that the epidermal barrier function is fully competent at birth [30,35], has been challenged by others who showed elevated TEWL in infants in comparison to adults [7,14,36]. TEWL in full-term newborns is generally below 10 g/m 2 /h, which is comparable to values in healthy adult skin [37]. Elevated TEWL values were demonstrated in the first 4 h after birth returning to normal values. This is suggestive of ongoing desiccation of the hyper-hydrated stratum corneum immediately after birth [38]. Comparative TEWL values of adults and infants in the post-neonatal period (older than 1 month) revealed no significant differences [39]. The GA influences the functional competence of the epidermal barrier function as a function of SC maturation. Preterm newborns showed elevated TEWL values compared to full-term babies [40] with an inverse correlation between GA and TEWL values. Interestingly, early after birth, small-for-gestational-age infants have lower TEWL values compared to appropriate-for-gestational-age babies born at the corresponding gestational age [41].
Anatomical variation of TEWL values was studied in 7 different regions of full-term babies [42]. TEWL was significantly elevated in the palms, soles and the volar forearm (close to the cubital fossa). A different study could not confirm these regional variations in TEWL values comparing the dorsa of the hand and foot [43] with the abdomen, buttock and forearm [44]. Anatomical site variations in the adaptation of the epidermal barrier have been described [45], together with ethnic-specific adaptation of TEWL values after birth [46,47].
There is limited data on gender influences on epidermal barrier function in infants. No differences were described for TEWL values regarding gender neither in full-term nor in preterm babies [40,42]. A difference between male and female babies was observed later in life. This difference is most likely related to hormonal divergences, which are not yet present at birth or early in childhood. Only the influence of maternal hormones can be seen clinically after birth, for example as acne neonatorum [48].

Hydration and water-handling properties of stratum corneum
A good correlation between SC hydration and epidermal barrier function (TEWL values) was detected in newborn and infant skin [43,49]. Following the initial period of evaporative desiccation right after birth, it is recognized that SC hydration of term-born babies is lower in the first days after birth compared to older children and adults [37,42,50,51]. Follow-up studies could demonstrate that SC hydration reaches a stable state between 2 weeks and 1 month after birth [50,51]. Dynamic hydration measurement revealed a diminishing water-holding capacity (in the sorptiondesorption test) during the neonatal period [51].
Infants with GA higher than 30 weeks had significantly lower SC hydration values than those born younger than the 30th week of gestation. [49]. These findings can partly be explained by the incomplete development of vernix caseosa (the so-called hydrophobic mantle) in premature babies [33,52]. SC hydration was compared in vernixretained and vernix-depleted skin areas at corresponding anatomical areas in full-term babies [53]. Higher hydration values were seen in vernix-retained regions, as well as significantly lower erythema grades and more acidic skin surface pH values. SC hydration is similar or even elevated in older infants (3-48 months PNA) compared to adults [1,7,39]. However, the actual water gradient and the water-holding properties differ significantly. A higher water concentration at the skin surface and within the upper 26 μm was measured in infants (3-12 months PNA) compared to adults [7]. A steeper SC water gradient was seen in infants. Lower levels of NMF measured by in vivo Raman Confocal Spectroscopy were documented in the infant epidermis. These findings can partly explain the faster water desorption in infants [7]. However, other factors than the NMF concentration (e.g. corneocyte maturation and SC lipids) are possible explanations for the faster and higher water absorption in infants. Other studies showed that baby skin had lower SC hygroscopicity (capability to bind water) compared to adults [39].
Significantly lower SC hydration values on the forehead, back and abdomen and higher values on forearms and palms were observed in the first postnatal days of life of term-born neonates compared to healthy adult volunteers [42]. Capacitance values (measure for SC hydration) between day 1 and 2 postpartum showed an increased SC hydration on palms and forearms, while lower values were assessed at the inguinal region [42]. The cheek of infants (3 and 6 months PNA) was significantly less hydrated than the cheeks of their mothers. These values were related to decreased skin surface lipids [37]. Such differences were not observed on the volar forearm. Climatic factors (e.g. wind, direct sun and air exposure) might be the origin of lower SC hydration in infant facial skin. However, young children are generally well protected from exogenous climatic stressors. No gender difference was observed in SC hydration for young children [50].

Acidic skin surface or the so-called 'acid mantle'
The so-called 'acid mantle' is a prerequisite for maintaining and adapting the anti-microbial, chemical and physical resistance of the skin [54,55]. Traditionally exogenous mechanisms of skin surface pH formation are known. But in addition, three endogenous pathways have been proposed to contribute to the formation of an acidic skin surface pH: secretory phospholipase A2 generates free fatty acids from phospholipids [56]; histidase-catalysed degradation of histidine [57]; and a non-energy-dependent sodium proton exchanger (NHE1) [58].
Skin acidification is essential for the epidermal barrier maturation and repair mechanisms, for example enzyme activity of βglucocerebrosidase and acidic sphingomyelinase (key enzymes in the extracellular processing of SC lipids) exhibit an acidic pH optimum [59]. Moreover, an increase in pH leads to the decay of these two enzymes by inducing serine protease activities [60,61]. Raising skin surface pH offers optimal conditions for the enzymatic action of kallikrein 5 (previously named SC trypsinlike enzyme) and kallikrein 7 (previously named SC chymotrypsin-like enzyme) [62,63]. A superbase-induced elevation of the SC pH resulted in a reversible increase of enzyme activity in a mammalian model [60]. The clinical observations of increased desquamation in the first days after birth can be associated with enhanced activity of these two enzymes in the more alkaline skin surface of newborn babies.
Right after birth, the skin surface pH of both full-term and preterm-born babies is elevated (less acidic) compared to older children or adults and decreases within the first weeks postpartum [39,50,64,65]. Prematurity and pH could not be directly linked. Gestational age did not influence the adaptation of surface pH after birth [64].
Independent of gestational age and birthweight, pH decreases steeply in the first days after birth and then more gradually in the rest of the neonatal period [50,64,65]. The mean pH value from six different body sites on the first day of life (full-term neonate) was 7.08, which represents significantly higher values than in adults (5.70) [42]. During the following day, a further decrease in surface pH was measured but was still significantly higher compared to adult values. The decrease in pH from day 3 to day 30 after birth was most accentuated on the volar forearm compared to the forehead, cheeks and buttocks [50]. The pH values remained at a stable level later in infancy (day 30 and day 90) for all tested skin sites. At later time points, skin surface acidity is similar to adult values [39].
Early after birth (day 1 and day 2) no differences in surface pH could be measured regarding anatomical sites [42]. Accordingly, pH did not differ significantly between sites early after birth (day 3) [50]. Later, at day 90, pH was higher on the cheek and buttock and lower on the forehead and volar forearm. The pH decrease in neonates was delayed in the diapered area (due to the semi-occluded, hyper-hydrated conditions) [51]. At the end of the first month, pH was significantly higher in the diapered area compared to more acidic non-occluded areas.
Low birthweight neonates females have less acidic skin surface pH than males [65]. Very low birthweight infants show higher pH values in males compared to female babies [64]. A different study showed no gender differences in pH neither in full-term neonates [50,66] nor later in infancy [1].

Sebum secretion
Sebum lipids are major constituents of Marchionini's protective cutaneous hydrolipid film concept, published in 1928. The sebaceous gland lipids are part of the nonspecific protective mechanisms of the skin barrier [67,68]. Sebum lipids contribute to vernix caseosa formation-the hydrophobic film covering the foetus in the third trimester. Vernix caseosa participates in the regulation of water/ electrolyte transport, thermoregulation, antioxidant protection and epidermal barrier development during foetal development [69][70][71].
In the first week after birth, an increase in sebum secretion is observed, reaching the level of adult excretion rates [72,73]. The sebum excretion rate of neonates and their mothers showed a good correlation [73]. In the following weeks and months, this correlation was lost confirming the role of transplacental hormonal stimuli for the sebaceous gland activity after birth. At age 6 months levels of sebum secretion were lower than after birth [72]. Sebum secretion remains relatively low and constant until pre-puberty when the hormonal maturation induces an increase in sebaceous gland activity, for example seen in acne patients [74].
Gender differences exist in the dynamics of sebum secretion in the first weeks after birth life [72]. At birth sebum secretion is lower in females, followed by a more pronounced increase (day 3-6 PNA) and a subsequent decrease [72].

Eccrine sweating
Both, thermal and emotional sweating have been evidenced in full-term neonates [75,76]. Preterm infants did not respond to thermal stimuli immediately after birth, however, at 13 days PNA thermal sweating was shown in all neonates [75]. Birth induced the development of thermal sweating. A relation between the intensity/extent of the sweat response and the GA was revealed [75]. Similarly, emotional sweating is more pronounced in full-term neonates, while in preterm newborns it is either weaker or absent [76,77].

Cutaneous blood flow and colour
Birth induces a series of modulations in cutaneous vascularization. They are related to the adaptation to the extra-uterine environment. Immature vasomotor control in premature vs. full-term infants was seen as a response to thermal stimulus on day 3 after birth [78].
The periodic oscillation of skin blood flux was examined in the first week after birth using laser Doppler flowmetry [79]. The oscillation frequencies in full-term neonates reached the lower range of adult values at the end of the first week. In contrast, premature infants showed values lower than full-term babies. High blood flow and limited potential of vasodilatation were measured in very low birthweight infants during the neonatal period [80]. These findings reflect an adaptation process of the cutaneous vasculature and are delayed in immature infants.
In preterm infants, the diameter of the blood vessels did not change significantly in the first month after birth [81]. Microvascular basal parameters did not dependent on gestational or postnatal age. No change in the vessel diameter was noted between days 1 and 5, and no difference could be measured between full-term and preterm infants [82]. Thus, a functional rather than anatomical immaturity of the cutaneous vasculature is responsible for the specific pattern of the cutaneous blood flow pattern in infancy.
Photometry was performed in preterm neonates were used to assess melanin skin concentrations. Higher melanin index on the areola skin was documented in infants with a GA between 34 and 37 weeks in comparison to those with a lower GA (24-34 weeks) [83].
In Figure 1 the dynamics of different functional skin parameters are summarized as a function of age (after birth).

SKIN MICROBIOME AND ANTI-MICROBIAL DEFENCE
Mammalian skin is not sterile and human skin is no exception. In contrast, its outermost layers host a variety of microorganisms-resident microflora: increasingly referred to as an important part of the integrity of the skin and other tissues, including an interaction with the immune system [84]. On the other hand, bacteria are crucial to infant immune system maturation via interactions with cutaneous epithelial and immune cells [85]. These effects on the immune systems are long-lasting and are called immune imprinting.
The evolution of the microbiome continues throughout the infantile period [11]. The skin microbiota of infants varies across individuals and is dependent on the analysed body site as well as on the infant's developmental stage [86]. Even the application of emollient cosmetics could result in increased skin-microbial diversity [87].
The alteration of the skin barrier milieu reflects the skin microbiome diversity and, therefore, leads to an altered immune response. It has been shown that commensal Staphylococci were less present in infants with AD at month 12 [88]. This could have a potential protective effect against disease development. Early colonization with commensal Staphylococci at 2 months after birth is related to a lower risk for AD development at age of 12 months [88]. Another example of the interaction on the 'skin barrier/microbiota/immune system' axis is certain findings in a mouse model: filaggrin deficiency and AD phenotype mice were compared to wild-type mice: There was a clear alteration in the skin microbiome of the filaggrin deficient mice with a higher presence of Corynebacteria and Streptococci [89]. Epidermal barrier integrity affects the skin microbiome composition as shown in humans [10]. Furthermore, diversity showed an inverse correlation with disease severity for lesional and non-lesional skin in AD subjects [90].
Bacteria residing in the skin belong generally to four phyla: Firmicutes (mainly genera Staphylococcus and Streptococcus), Bacteroidetes, Proteobacteria and Actinobacteria [84,91]. Anatomical site and age variations have been shown in skin microbiota [11]. The amount of Staphylococcus species is higher on neonatal skin in comparison to adults and with a skin microbiome of newborns resembling the microbiome of moist adult skin sites [17]. Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes are mainly predominant, and an ongoing adaptation of skin microbiota continues in childhood [11,17]. In addition, ethnic and geographic skin microbiome in infants has been witnessed between Estonian and Finnish children, and the role of the genus Acinetobacter in the sensitization pattern between both populations has been suggested [92].
Term neonates show undifferentiated skin microbiomes across body sites [11]. Subsequently, the surface microbiota adapts to the extra-uterine environment and their inter-site diversity reaches one in adults [11,93]. Infants delivered vaginally show skin microbiome dominated by Lactobacillus, Prevotella and Sneathia species, while the skin of Caesarean section-born babies displays bacterial microbiota similar to their mother's cutaneous microbiota with a predominance of Staphylococcus, Corynebacterium and Propionibacterium species [93]. Delivery mode, however, is not affecting the infant oral microbiota beyond 4 weeks of age, while oral infant microbiota continues to develop until the age of 1 year [94]. The effect of the mode of delivery seems to dissolve by the age of 1 month [91,95]. An increased alpha diversity with age was evidenced in children <10 years, independent from body site [96]. These data reflect the ongoing adaptation of the skin microbiome in childhood. Gestational age and less antibiotic use are correlated to greater skin bacterial diversity [91,97,98]. Preterm infants have an increased abundance of Staphylococcus, Corynebacterium (phylum Actinobacteria) and Prevotella (phylum Bacteroidetes) [91,98].
The skin microbiome was studied in infants with diaper dermatitis showing higher microbial diversity in patients when compared to healthy controls [99]. The predominant species in diaper dermatitis patients belonged to the groups of Proteobacteria, Enterococcus, Erwinia, Pseudomonas and S. aureus, with the recovery of S. epidermidis after emollient treatment [99].
In AD infants, the predominance of Bacteroidetes and Fusobacterium, and Prevotella showed an inverse correlation to disease severity [100]. Reduction in Staphylococcus aureus and normalization towards a diversification of the skin microbiota correlated to the therapeutic improvement of AD.
Skin mycobiome was studied in infants younger than 6 months, showing a decrease in skin fungal diversity with age with the Malassezia being the predominant genus, and M. globose-the most common species [101]. The interindividual variability in skin mycobiome was higher than the inter-anatomical site variations in the same subject.
In general, the levels of anti-microbial peptides (AMPs), key players in innate immunity secreted mainly by keratinocytes (lamellar bodies) and immune cells (but also by resident microflora), are lower in infants than in adults [102,103]. In contrast to total AMPs count, lactoferrin and lysozyme were higher in newborns [103]. AMPs are present at birth and have also been detected in vernix caseosa [104]. The clinical implication of these findings could be that an anti-microbial defence mechanism is already available at birth accomplished by AMPs. Another crucial element in innate immunity, toll-like receptors (TLRs), undergo differentiation after birth. A complete similarity was witnessed in the TLRs expression of children and adults [105]. Cutaneous prenatal TLRs 1-5 and infant TLRs 1 and 3 were increased in comparison to adults. In addition, TLRs agonists stimulated more pronounced secretion of CXCL8/IL8, CXCL10/IP-10 and TNFα in foetal and neonatal keratinocytes in comparison to adult samples [105].

CONCLUSION
The human skin barrier adapts to the extra-uterine gaseous environment after birth. This ongoing process can be witnessed on a structural, biochemical and functional level ( Figure 2). Despite that human skin accomplishes competent barrier function under basal conditions after birth, structural and functional parameters such as corneocyte size and distribution, skin hydration and surface acidity, microvasculature and skin-microbial flora interactions continue to evolve after birth.

ACKNO WLE DGE MENTS
None Open Access funding enabled and organized by Projekt DEAL. F I G U R E 2 A schematic overview on the principle differences between newborn and adult skin.