These studies reveal decreased levels of proteins related to the skin barrier (filaggrin-2, corneodesmosin, desmoglein-1, desmocollin-1, and transglutaminase-3) and generation of natural moisturizing factor (arginase-1, caspase-14, gamma-glutamyl cyclotransferase) in lesional versus nonlesional sites of EH+ and EH− AD patients. Epidermal fatty acid binding protein was expressed at significantly higher levels in patients with methicillin resistant Staphylococcus aureus as compared to patients with methicillin sensitive Staphylococcus aureus. No significant differences were found between AD patients with and without a history of eczema herpeticum, but many proteins neared the significance threshold, notably filaggrin-2.
Recent studies indicate that defects in skin barrier proteins are highly associated with the development of AD. Loss of function mutations in filaggrin can be found in approximately 20% of AD patients.15
Filaggrin, a member of the fused S100 family of S100 Ca2+
-binding proteins, is synthesized in the granular layer as a large 400 kDa precursor termed profilaggrin.16
Profilaggrin is stored within keratohyalin granules in the granular layer;17
as calcium levels rise during differentiation, it undergoes extensive processing including dephosphorylation and cleavage into filaggrin monomers.17
In the cornified layer, transglutaminases crosslink filaggrin to keratin 1 and 10 to form the insoluble keratin matrix crucial to the development of the skin barrier (see ).18
Next, the cross-linked filaggrin monomers undergo further post-translation modification (deimination/citrullination) via the calcium dependent enzyme peptidylarginine deiminase (PAD).18
This deimination results in disruption of the filaggrin/keratin crosslinking, setting the stage for filaggrin degradation into NMF. NMF refers to a mixture of primarily filaggrin derived hygroscopic amino acids including arginine, glutamine, and histidine and their derivatives citrulline/urea, PCA (2-pyrrolidone-5-carboxylic acid), and UCA (urocanic acid), respectively (see ).3
a) Generation of Natural Moisturizing Factor
Numerous enzymes are involved in the processing of profilaggrin to filaggrin to NMF as reviewed by Sandilands et al
and Candi et al
In this exploratory study, three filaggrin/NMF processing enzymes were found to be expressed at significantly lower levels in EH+ and/or EH− lesional AD skin as compared to nonlesional AD skin (caspase-14, gamma glutamylcyclotransferase (GGCT), and arginase-1). Additionally, bleomycin hydrolase (BH), while not statistically significant, showed a trend towards lower expression in EH+/EH− lesional vs nonlesional skin. Caspase-14 is an enzyme required for the processing of deiminated filaggrin, and homozygous null mice lacking caspase-14 display mild barrier defects characterized by increased transepidermal water loss, decreased SC hydration and abnormal filaggrin degradation.19
The neutral cysteine protease bleomycin hydrolase (BH) is important in the final breakdown of partially processed and deiminated filaggrin peptides into amino acids which are components of the NMF.20
Gamma-glutamyl cyclotransferase (GGCT) catalyzes the formation of pyroglutamic acid or PCA which is the most abundant NMF found in the stratum corneum.21
Arginase-1, an enzyme in the urea cycle, hydrolyzes L-arginine into L-ornithine and urea.22
Arginine is a significant amino acid component of filaggrin18
and is released upon filaggrin degradation. Decreased expression of arginase-1 may decrease urea generation, a hygroscopic component of the NMF.23
As a whole, these data indicate altered filaggrin processing in lesional skin which may further exacerbate the disease process via abnormal corneocyte development and a decrease in the amount of NMF which is crucial to skin hydration. Restoration of NMF components using creams and moisturizers containing urea or PCA has been shown to alleviate the symptoms of AD and/or reduce the risk of relapse as reviewed by Loden.24
The ability of these NMF-based creams to restore the skin barrier further highlights the critical role of NMFs in skin barrier integrity.
In addition to lower expression of enzymes involved in NMF generation, our current work revealed lower expression of three proteins directly linked to the skin barrier and corneodesmosome structure. Corneodesmosomes, comprised of desmoglein-1, desmocollin-1, and corneodesmosin, bind keratins to the cellular membrane and serve to tightly attach adjacent corneocytes ().18
Through a tightly controlled process, corneodesmosomes are proteolytically degraded in the uppermost layers of the stratum corneum to allow desquamation. The remaining keratins are covalently attached to the cell envelope and provide mechanical resistance.25
Simultaneously, the cytosolic enzyme transglutaminase-3 (protein-glutamine γ-glutamyltransferase E or TG3) mediates the crosslinking of loricrin to small proline rich proteins; this complex further reinforces the cell membrane (). The significantly lower expression of desmocollin-1, desmoglein-1, corneodesmosin, and TG3 in lesional skin could be indicative of inappropriate desquamation26
or abnormal differentiation3
both of which have been found in AD.13
Filaggrin-2 (flg-2) is one of five genes in the S100 fused-type protein gene cluster.27,28
It is closely homologous to filaggrin, but the precise role of flg-2 in skin biology is unknown. The lower expression of flg-2 in both EH+/EH− nonlesional skin vs non-atopic nonlesional skin and in EH− lesional vs nonlesional skin indicate a potential role in the maintenance of the skin barrier. Furthermore, there was a trend towards lower levels of flg-2 in EH+ NL as compared to EH− NL (P
=0.02) skin. Unpublished work reported in a patent by Jens-Michael Schroder indicates potent antimicrobial activity of the C-terminal of flg-2 against the soil bacterium, Pseudomonas
Our observed lower expression of flg-2 in EH+ nonlesional skin is consistent with previous work showing an association of impaired skin barrier and decreased antimicrobial activity in subjects prone to EH.30,31
Further work needs to be done on this potentially important skin barrier protein and antimicrobial peptide as a biomarker distinguishing EH+ vs EH− subjects.
In our present study, e-fabp was elevated in MRSA lesional vs MSSA lesional and MRSA lesional vs. no S. aureus
lesional. Elevated levels of fatty acid binding protein in AD patients have been reported in other studies,14,32
but none evaluated concurrent S. aureus
colonization status. Fatty acid binding proteins are abundant intracellular proteins that bind and transport otherwise insoluble long-chain fatty acids.33
E-fabp is found in the basal and granular cell layers in normal human skin34
and appears to be essential for normal keratinocyte differentiation.35
It has been proposed that fatty acid binding proteins may serve as master regulators of inflammatory and metabolic signaling pathways.36
In support of this, e-fabp has been shown to bind and stabilize leukotriene A4
) and may modulate the production or metabolism of bioactive eicosanoids which have been found in the urine of AD patients.37,38
In addition to a potential role in eicosanoid signaling, e-fabp may also serve as an antioxidant protein and has been shown to bind 4-hydroxynonenal, a highly reactive aldehyde by-product of lipid peroxidation.39
Increased levels of urinary oxidative stress markers have been found in AD patients40
as well as direct evidence of oxidative stress in the stratum corneum in AD.41
It is possible that oxidative stress may induce e-fabp, and this may be exacerbated in AD patients with MRSA infection. Additionally, liver fabp has been shown to be a critical host factor for malaria42
and increases the intracellular growth of chlamydia.43
Further studies are needed to determine whether e-fabp may promote S. aureus
growth or serve as a host factor for infection.
In conclusion, we have found lower expression of skin barrier proteins in lesional skin of AD patients. These proteins are involved in the generation of the NMF, corneodesmosomes, and antimicrobial host defense. These changes may reflect defective differentiation of corneocytes in AD and promote susceptibility to skin infection. The findings presented here support recent work highlighting broad defects in epidermal cornification in AD.13
In addition, increased e-fabp levels in MRSA infected AD patients may indicate aberrant eicosanoid signaling, oxidative stress, or e-fabp may serve as a host factor for S. aureus
- The lower levels of four enzymes involved in the generation of the natural moisturizing factor in lesional atopic dermatitis skin are a novel finding. This defect could perpetuate the dry skin cycle and predispose patients to infection.
- The lower levels of key skin barrier proteins involved in the generation of corneodesmosomes may result in decreased corneocyte adhesion. The lower levels of filaggrin-2 in atopic dermatitis lesions may indicate a skin barrier defect or a decrease in antimicrobial peptides.
- The higher levels of epidermal fatty acid binding protein in patients colonized with methicillin-resistant Staphylococcus aureus as compared to patients colonized with methicillin sensitive Staphylococcus aureus or no S. aureus may represent a protective mechanism to elevated oxidative stress or may perpetuate the inflammatory response.