Журнал «Здоровье ребенка» 2 (45) 2013

For a long time, allergic diseases have been considered as primarily immunologic disorders. As a consequence, research and drug development focused on modifying the Th2 effector phase. Over the last decade, there has been a shift in our thinking, with the epithelium now recognized as a critical player in the development of allergic sensitization (De Benedetto et al., 2012). In particular, compelling data from human and mouse studies have implicated skin barrier impairment as an indispensable event in allergen sensitization.

The specific pathways connecting epidermal barrier disruption to allergen sensitization are beginning to be elucidated. The overriding hypothesis is that epidermal disruption would allow skin resident antigen presenting cells such as Langerhans cells or dendritic cells to capture environmental antigens. Several case–control studies have also demonstrated strong association between mutations in filaggrin gene (FLG) and early onset of atopic dermatitis (AD), disease severity, AD-related asthma and greater allergen sensitization. Interestingly, several studies have shown FLG mutations confer a substantial risk for other atopic disorders recognized as Th2 polarized diseases including allergic rhinitis, IgEmediated peanut allergy, and eosinophilic esophagitis.

The murine models of epicutaneous allergen inflamation induce skin lesions like in atopic skin. The sensitized mice have increased scratching behavior, thickened epidermis, inflammatory infiltration of CD4+ T cells and eosinophils, expression of Th2 cytokines, with modest increases in IFNγ, Th2 chemokines such as eotaxin and the Th2 adjuvant, TSLP and allergen-specific IgE. Additionally, these mice developed airway hyper-responsiveness to methacholine and eosinophilia after a single dose of inhalation of the allergen. This demonstrates that antigen education epicutaneously on barrier-disrupted skin is sufficient to elicit systemic Th2 allergic inflammation in a distant organ such as the lower airways and esophagus following relevant immune response.

 To demonstrate the importance of the route of immunization (e.g. sensitization phase), it was evaluated whether the immunologic response differed if the allergen (e.g. peanut protein) was applied on disrupted epidermis (24 hours after tape stripping) or injected subcutaneously with complete Freund’s adjuvant. The epicutaneous route generated a Th2 immune response in draining lymph nodes and spleen (higher IL4 and antigen-specific IgE and lower IFNγ, IL10 and IgG2a) in contrast to the subcutaneous route, which induced a Th1 immune response (high IFNγ and IgG2a and low IL4 and antigen-specific IgE) ( Sandilands, 2009).

The key function of the skin is a protective barrier between the host organism and its external environment, minimising water loss and preventing the entry of microorganisms and allergens. Terminal differentiation of keratinocytes results in the formation of a densely packed and extensively crosslinked lipid-protein matrix, which forms an impenetrable barrier (known as the stratum corneum) that is the uppermost (cornified) layer of the epidermis.

A defective skin barrier is a key feature of the chronic inflammatory skin disease atopic dermatitis. It was demonstrated that the late epidermal differentiation protein filaggrin has a primary role in skin barrier function and that null mutations within the FLG gene strongly predisposes individuals not only to atopic dermatitis, but also to associated secondary allergic diseases such as asthma. Filaggrin is synthesised as a giant insoluble precursor protein, profilaggrin.

Profilaggrin forms the major component of the keratohyalin granules that are visible by light microscopy within the granular layer of the epidermis. The profilaggrin molecule is composed of an N-terminal domain which has calcium-binding and nuclear localization components, followed by 10, 11 or 12 nearly identical filaggrin repeats which have keratinbinding properties, and a C-terminal domain of unknown function (Sandilands et al., 2009). Loss-of-function mutations within FLG exon 3 result in a truncated profilaggrin molecule which lacks the C-terminus, resulting in an almost complete absence of filaggrin monomers.

The proprotein itself has no keratin-binding activity but, during the later stages of epidermal terminal differentiation, profilaggrin is dephosphorylated and proteolysed into multiple filaggrin monomer. The proteolytic conversion of profilaggrin to multiple copies of monomeric filaggrin during the transition from a granular cell to a cornified cell is a tightly controlled multistep process that involves dephosphorylation by one or more phosphatases and site-specific proteolysis.

 Monomeric filaggrin binds to keratins 1 and 10 and other intermediate filament proteins within the keratinocyte cytoskeleton to form tight bundles, facilitating the collapse and flattening of cells in the outermost stratum corneum to produce squames. Loss-of-function mutations in FLG may be associated with disorganized keratin filaments, impaired lamellar body loading and abnormal architecture of the lamellar bilayer (Gruber et al., 2011).

Breakdown of filaggrin into hygroscopic free amino acids and their derivatives, such as pyrrolidone carboxylic acid (PCA) and trans-urocanic acid (trans-UCA), are the major contributor to the ‘natural moisturising factor’ (NMF) that is produced within the stratum corneum. NMF is responsible for maintaining the pH gradient of the epidermis, skin hydration and water retention within the stratum. Free amino acids have a well-known antimicrobial effect and there is evidence that filaggrin breakdown products at physiological concentrations demonstrate an inhibitory effect on the growth of Staphyloccus aureus. An acidic pH within the stratum corneum is also important for the functional activity of enzymes involved in ceramide metabolism (Fluhr et al., 2010). FLG null mutations are associated with lower levels of hygroscopic amino acids in the stratum corneum, and there is a concomitant increase in transepidermal water loss. It is well known that the magnitude of the barrier defect in AD, as measured by transepidermal water loss (TEWL) at nonlesional sites, correlates with disease severity and serum IgE. AD infants with two or more positive allergen patch tests had higher TEWL than infants with one or no positive tests (Boralevi et al., 2008).

Evidence supporting a role for filaggrin in genetic susceptibility to eczema came from genome-wide association studies, which had identified statistically significant genetic linkage with polymorphic markers within the epidermal differentiation complex on chromosome 1q21, where the FLG gene is located. R501X and 2282del4 are the two most common mutations and they have consistently shown significant association with atopic eczema across the continent.

The strong and highly significant association of FLG null mutations with atopic eczema has subsequently been replicated in over 20 independent studies, including case/ control studies, family studies and unselected population cohorts. Two recent meta-analyses of these data have estimated the odds ratio of developing atopic eczema to be 4.78 (van den Oord and Sheikh, 2009) and 3.12 (Rodriguez et al., 2009) in association with FLG null genotype. The eczema sub-phenotype that is most strongly associated with FLG null mutations is that of early onset, severe and persistent disease (Brown et al., 2012) and with the associated ‘extrinsic’ features of raised total IgE and allergic sensitization (Weidinger et al., 2007).

In studies of filaggrin-deficient children, increased transepidermal water loss along with development of specific IgE antibody to dust mite and cat was found with asthma.

Each of the reported FLG null mutations therefore have an equivalent molecular biological effect, since they each produce biochemically unstable truncated profilaggrin which cannot be processed to release functional filaggrin. Future research into the filaggrin molecule and defects in the filaggrin gene holds great potential to improve the care of atopic eczema patients and ultimately to prevent the development of atopic disease.