We report data demonstrating the importance of the skin in the development of fetal inflammation. Inflammation of the fetal and maternal tissues following microbiological invasion of the uterine cavity is one of the most important causes of preterm birth. Between 25% and 40% of all preterm births (delivery before 37 weeks gestation) are associated with intrauterine infection.9
By exposing the fetal and maternal tissues to E. coli
LPS, a number of investigators have studied the evolution and effects of intrauterine inflammation in animal models including mice, rabbits, nonhuman primates, and sheep.14–17
We and others have clearly demonstrated that intra-amniotic administration of LPS induces chorioamnionitis, inflammation, and maturation of the fetal lung in conjunction with elevated levels of fetal and amniotic cytokine/chemokine expression. Despite our appreciation of the role played by the innate immune system and inflammation in the premature induction of parturition, there remains a number of unresolved questions in relation to the timing and origin of the inflammatory stimuli that drive these processes.
In this study, we demonstrate the importance of the skin in the development of fetal inflammation. The fetal skin is exposed in its entirety to the amniotic environment and thus any colonizing microorganisms. Previous histological investigation of the developing fetal skin in humans demonstrated that the outer cornified stratum corneum, which plays an important barrier function by virtue of its high lipid and keratin composition, is largely absent in the first 2 trimesters of gestation.20
We suggest that even a low-grade inflammatory response in the fetal skin is likely to have significant systemic impact on the fetus due to the skin possessing both a large net surface area and ample vascularization.
Keratinocytes constitute approximately 95% of the adult epidermis; accordingly it is not unreasonable that keratinocytes are significant constituent of the fetal skin. Our analysis of fetal epidermis at 124 days gestation using antibodies against involucrin (a keratinocyte-specific marker of differentiation) demonstrated robust epidermal staining (). Adult human keratinocytes respond to E. coli
LPS via TLR4-driven signaling and express a diverse complement of innate immunoreceptors.30
We established a reliable system for culturing fetal epidermal keratinocytes and demonstrated that these cells possess the capacity to respond to LPS in vitro (). Interleukin 1β, IL-6, and TNF-α constitute a triumvirate of acute-phase proinflammatory cytokines that drive the development of an inflammatory response following tissue injury or exposure to a host of microbial pathogens.31
Analysis of mRNA transcript expression for these cytokines and IL-8 demonstrated significant increases as early as 100 minutes after exposure. Elevated expression of cytokine transcripts generally persisted for up to 4 hours. Initial studies of IL-8 demonstrated its importance as a neutrophil chemotactant, and it has subsequently been suggested that this cytokine plays a role in the chemotaxis of both basophils and activated eosinophils.32,33
Interleukin 8 is also involved in maturation of the inflammatory response of neutrophils; specifically the induction of morphological alterations, increased expression of adhesion molecules (including CD18) enhanced tissue migration (especially into epidermal and dermal tissues), increased superoxide and lipid production along with lysosomal enzyme release.34,35
This acute increase in expression followed by return to baseline is in concordance with our own studies and those of other investigators conducted in the fetal lung and other fetal endothelial tissues.19
Having established that the fetal keratinocyte is able to mount a proinflammatory response to a TLR4 agonist, we sought to study the ability of the fetal skin to respond to E. coli LPS in vivo. By either completely exposing the fetus to LPS via intra-amniotic injection or restricting LPS exposure to the respiratory tract following surgical ligation of the trachea, we were able to demonstrate that the fetal skin expressed elevated levels of IL-1β and IL-8, 48 hours after treatment but only when directly exposed to LPS. This observation has 2 important implications for the skin as a mediator of fetal inflammation. First, it demonstrates agreement between our in vitro and in vivo studies and suggests that the fetal skin has the ability to respond, in a proinflammatory manner, to the presence of TLR4 agonist in the amniotic fluid. Second, the absence of an inflammatory response (as detected by IL-1β and IL-8 expression) in animals where LPS exposure was restricted to the lungs provides the first evidence that the presence of bacterial agonist in the amniotic milieu is both sufficient and necessary for the fetal skin to generate a localized inflammatory response.
We previously demonstrated that localized increases in cytokine expression in lung tissue are rapidly accompanied by cellular infiltration as part of the developing inflammatory response.19
In parallel, we observed an inflammatory response to LPS in the fetal skin characterized by the extensive recruitment of CD18 positive cells following direct exposure to LPS. The results of this investigation clearly show a marked elevation in CD18 staining in the dermis of fetal skin directly exposed to LPS via intra-amniotic administration. The administration of saline (irrespective of route) or intratracheal LPS failed to induce an alteration in CD18 staining. From both our light and immunofluorescence analyses, we can conclude that direct exposure of the fetal skin to LPS induces an epidermal infiltrate and that activated neutrophils comprise a significant component of this infliltrate. Therefore, the fetal skin is capable of generating a localized de novo inflammatory response. Activated neutrophil infiltration is a well-characterized response (most notably in epidermal and dermal tissues) of proinflammatory cytokine expression, in particular in response to localized increases in the expression of IL-8.
A further well-characterized effect of LPS-driven immune stimulation is the induction of alterations in TLR-4 receptor expression. TLR-4 is the primary innate immune receptor for gram-negative LPS. TLR4 activation occurs via the formation of a complex of adaptor proteins including LPS-binding protein CD14 and MD2 with LPS.36
Intracellular adaptor proteins MyD88 and Mal regulate cytokine/chemokine expression via Nuclear factor κB (NFκB) signaling and interferon 1 expression via the Trif Related Adaptor Molecule (TRAM) and TIR domain-containing adaptor inducing IFN (TRIF) signaling pathways.37,38
Several investigators reported increases in TLR expression following exposure of cells to LPS or inflammation.39
Our immunohistological analyses identified increased spatial distribution and intensity of TLR4 staining of fetal skin in animals directly exposed to LPS relative to saline control alone. Interestingly, it appears that a number of epidermal appendages adjacent to tissues directly exposed to LPS also demonstrate enhanced TLR4 expression. These images suggest that either direct LPS exposure itself or the resultant, locally acting inflammation increases TLR4 expression, demonstrating that the fetal skin is able to mount a proinflammatory response following direct exposure to LPS ().
Spontaneous preterm birth is likely the common “end point” of inflammation subsequent to intrauterine inflammation. Infection of the amniotic cavity results in the production of proinflammatory cytokines and chemokines that, through a variety of downstream mediators including vascular endothelial growth factor (VEGF), matrix metalloproteinases, and prostaglandins, induce degradation of the fetal membranes, cervical remodeling, and bring uterine quiescence to an end. Both “preterm birth” and “infection-derived inflammation” are enormously broad terms covering an extended range of interconnected inflammatory mediators and signaling systems activated in response to an as yet loosely defined (but most likely large) number of contributing microorganisms potentially acting from before conception up to 36 weeks ofGA.
One of the very important questions that remains unanswered is the relative importance of maternal versus fetal inflammation and, more specifically, which tissues (chorioamnion/placenta/fetal lung/fetal skin) are responsible for generating the numerous cytokines and chemokines that initiate and drive the pro-parturition inflammatory processes we know to be causative of preterm birth. To this end, we hypothesize that the developing fetal skin may play a role in the induction of fetal inflammation in response to intrauterine inflammation. We have demonstrated that the fetal skin is capable of generating an inflammatory response in utero to E. coli LPS. We show that that this response involves proinflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) expression, the recruitment of activated neutrophils, and modulation of TLR4 expression and requires direct exposure to bacterial agonist.
Having demonstrated its ability to respond to bacterial agonist, we propose that the fetal skin is an important site of the inflammation that also contributes to the FIRS. The uncornified nature of the developing fetal skin at early GAs is likely to render it permissive to the transepidermal passage of compounds (including cytokines and chemokines) from the fetus into the amniotic environment.
There are a number of clinical implications of the current study. Using proteomic approaches, investigators have recently demonstrated that desquamated skin epidermal cells from neonates collected by an adhesive tape had increased expression of several chemokines/cytokines.40
In the context of chorioamnionitis-induced fetal inflammatory response, skin inflammation may contribute to both products of inflammation in the amniotic fluid and fetal blood. Additionally, skin inflammation may further compromise the already poor barrier function in the preterm skin. Thus, infants with exposure to chorioamnionitis may have increased predispostion to nosocomial infection due to potential alterations in skin barrier function. Further extrapolation of the data presented in this study is presently hindered by our limited understanding of the effect/effects of inflammation on the maturation of the fetal skin, putative induction of systemic inflammation, and the degree of concordance between the developmental trajectories of human and ovine skin in utero. In addition to the above, we suggest that the functional maturation of the fetal skin (with specific reference to the development of a mature stratum corneum following programmed terminal differentiation of epidermal keratinocytes) at differing GAs may affect the proinflammatory role played by the fetal skin in the setting of an in utero infection.
Despite these acknowledged limitations, we suggest that the data presented herein indicates for the first time that the fetal skin may be an important source of the both fetal inflammation and proinflammatory compounds that then act on the membranes, cervix, and uterus to ultimately induce preterm labor.