Human atopic dermatitis (AD) is increasingly viewed as a primary barrier disorder, based upon recent data from multiple populations that have demonstrated an association with mutations in FLG
, the gene encoding the stratum corneum structural protein, filaggrin (FLG). Yet, while these molecular genetic studies have shifted views of AD pathogenesis towards a new ‘outside-to-inside’ pathogenic paradigm 1, 2
, they leave unanswered mechanistic questions of how FLG-deficiency leads not only to a barrier abnormality, but also to an inflammatory phenotype in AD. Because such questions are difficult to address in the complex, multifactorial environment of human disease, we addressed these issues in an animal model of flg deficiency, the flaky tail (ft/ft) mouse. Recently, these mice were shown to carry a frameshift mutation with one base pair deletion (5303delA) in exon3 of flg 24
, which mimics two common, distal mutations in human AD (i.e., R2447X in repeat 7 and 1033del4 in repeat 10) 6, 28
In our examination of ft/ft mice, we first confirm that flg deficiency alone suffices to induce a barrier abnormality and low-grade inflammation under basal conditions, as shown recently for ichthyosis vulgaris 50
. We also provide subcellular pathogenic insights, showing that despite its intracellular localization, flg deficiency results in enhanced paracellular permeability to both intradermally and epicutaneously applied water-soluble tracers, which do not penetrate +/+ stratum corneum. Movement of these low-molecular weight, water-soluble tracers through the stratum corneum interstices likely reflects the pathway for hapten ingress in AD, since similarly-sized, water-soluble antigens, such as nickel, more frequently induce allergic contact dermatitis in humans with AD 51
. However, since our ft/ft mice were not on a homogenous C578BL6 background, but also express a hair phenotype (matted), we cannot eliminate the possibility that some of our observations could have been influenced by the concurrent matted
mutation. Nevertheless, the recent work by Fallon, et al 24
shows enhanced antigen ingress in mice with the same flg
mutation, but no matted in their background. Thus, flg deficiency alone likely explains the barrier abnormality that we demonstrate here in flaky tail mice.
Localizing the barrier defect in FLG deficiency to the extracellular matrix is an important first step in understanding AD pathogenesis. Yet, a key question remains unanswered: how can a defect in an intracellular
protein, such as FLG, yield a more-porous extracellular
pathway? FLG has two putative functions: 1) mediation of stratum corneum hydration through humidity-dependent proteolysis of FLG into its constituent amino acids and their deiminated products (e.g., histidine → trans-urocanic acid)18, 52
; and 2) keratin intermediate filament compaction within the corneocyte cytosol 19, 53
. Examination of either of these roles does not immediately suggest a mechanism by which FLG deficiency could lead to increased extracellular
permeability. Potential clues are provided by other inherited defects of intracellular corneocyte proteins that also provoke increased paracellular permeability, but by divergent mechanisms 54
. In one example, in K1/10-deficient epidermolytic hyperkeratosis, dominant-negative pairing of the mutant protein disrupts the cytoskeleton, impairing lamellar body secretion 32
. In a second, alternate mechanism, both loricrin keratoderma and transglutaminase 1-deficient lamellar ichthyosis yield an abnormal corneocyte scaffold that disrupts the supramolecular organization of the extracellular lamellar bilayers 36, 37
. A third mechanism, mutations in the V1 subdomain of K1 that produce palmar plantar keratoderma, provokes detachment of keratin filaments from the cornified envelope 55
. This defect could link an abnormally-collapsed corneocyte to an extracellular defect, yet frozen sections of flg-deficient stratum corneum display corneocytes of normal shape and dimensions (Elias, unpublished observations). Our ultrastructural studies on ft/ft mice show a partial failure of lamellar body secretion, as shown previously for non-genotyped human AD 56
. Likewise, our preliminary studies in genotyped ichthyosis vulgaris +/− AD patients show a similar blockade in lamellar body secretion 50
. Thus, analogous to K1/10-deficient epidermolytic hyperkeratosis 32
, unprocessed profilaggrin might impede lamellar body secretion (op. cit.) (suppl. Fig. 2
). This mechanism likely also pertains to human AD associated with distal FLG
mutations, in which residual profilaggrin expression is seen, but processed FLG is reduced 28
. Yet, it is not clear how this mechanism might apply to those cases of AD caused by more proximal FLG
mutations, which instead decrease both profilaggrin and FLG. A fourth mechanism, which could apply to both categories of FLG mutations, could be linked to decreased generation of acidic metabolites of FLG 1, 2 suppl. Fig. 6
). Deficiency in these polycarboxylic acids could result in a net increase in the pH of the stratum corneum, which in turn could activate neutral-pH-dependent kallikreins, with a variety of negative, downstream consequences for the barrier 1,2
. Such a pH-dependent mechanism could also lead to deactivation of two key ceramide-generating enzymes, acidic sphingomyelinase and β-glucocerebrosidase (suppl. Fig. 6
. This mechanism may not, however, be operative in ft/ft mice, because the surface pH of these mice remains normal, perhaps due to compensatory upregulation of alternate, endogenous acidifying mechanisms [rev. in 58
], such as solute carrier family 9 (sodium-hydrogen exchanger, 1 (slc9a) (old symbol NHE1) 59
and/or secretory phospholipase A2 60
. Nonetheless, surface pH measurements do not always reflect pH alterations within specific microdomains of the stratum corneum 59
. We did not specifically examine such potential, compensatory mechanisms in this study.
While our studies demonstrate the increased movement of topically-applied, water soluble compounds through the stratum corneum in ft/ft mice, the movement of water from inside to outside through the stratum corneum (TEWL) is not markedly altered in young ft/ft mice, becoming only slightly elevated in older ft/ft mice in comparison to older +/+ mice. The development of a barrier abnormality with aging of ft/ft mice could reflect the added stress from a steeper water gradient across the SC of older animals reflecting the observed decrease in SC hydration (). Alternatively or additionally, it could reflect age-related differences in permeability barrier homeostasis 44, 61
. While the explanation for the discordance in TEWL rates vs. xenobiote penetration is not clear, it could reflect differences in the thresholds for the barriers to water loss vs. xenobiote ingress/egress, respectively. Pertinently, to increase TEWL levels by tape stripping, multiple layers of the stratum corneum must be removed 62
. Thus, there appears to be substantial redundancy, with only a relatively small amount of stratum corneum required to maintain a normal TEWL. Conversely, the structural basis for inhibiting water movement from inside to outside (i.e., TEWL) could differ from the structural basis for inhibiting the egress or ingress of somewhat larger, water-soluble compounds. Since there are several possible mechanisms that could explain how FLG deficiency leads to a marked abnormality in xenobiote permeation, without altering water loss, additional studies will be required to distinguish among these alternatives.
Having established that FLG deficiency results in an extracellular barrier defect, we next addressed a question of key functional significance; namely, does this barrier defect suffice to alter thresholds to the development of inflammation from epicutaneously-applied irritants and haptens? While FLG
mutations are associated with the inflammatory disease, AD (op. cit.), the relationship between FLG deficiency and inflammation is obscured by the association of the same FLG
mutations with the supposedly noninflammatory disorder of cornification, ichthyosis vulgaris (IV). Additional stressors to the barrier could be required to provoke human AD 1, 2
, because human skin displays an inherently more competent barrier 63–65
(see below). Nevertheless, the presumption that IV is non-inflammatory should now be re-examined, in light of our observations, and those of Fallon, et al. 24
, which show that flg deficiency alone results in epidermal hyperplasia, low-grade inflammation, and elevated IgE levels in otherwise normal mice.
In order to ascertain whether FLG deficiency alone leads to increased susceptibility to inflammatory dermatoses, we employed previously described methods for eliciting irritant and acute allergic contact dermatitis 38, 47
, as well as an AD-like mouse model 49
, in ft/ft vs. +/+ mice. Our results show that FLG deficiency alone lowers thresholds to inflammation following epicutaneous applications of either irritants or haptens. Most importantly, we demonstrate development of an AD-like phenotype following applications of reduced concentrations of haptens, which do not produce th2-dominant inflammation in +/+ mice. The AD-like phenotype included increased CRTH2-positive cells, decreased immunostaining for mBD3 (the murine homologue of hBD2), and elevated serum IgE levels. Similarly, antigen-challenged mice, with the same flg
mutation as ft/ft mice, also display multiple features of th2 inflammation 24
. Interestingly, these flg-deficient mice reportedly did not develop significantly higher IgE levels24
, likely reflecting a less severe phenotype due to differences in bioavailability of our hapten vs. the complete antigen employed by Fallon, et al. 24
. Finally, the apparent reduction in mBD3 expression could reflect downstream down-regulation by th2 cytokines 16
Notably, inflammation can be induced in flg-deficient mice, without a requirement for additional, acquired stressors, such as reduced ambient humidity, high pH surfactants, and/or increased psychological stress, conditions that are known to both further compromise barrier function, and to precipitate AD in humans 1–3
. This leaves unanswered, however, the question of why many IV patients, including some with double-allele FLG
mutations, do not display a concomitant AD phenotype 25, 26
. Two alternative or perhaps coincident explanations could explain this apparent paradox. First, it is possible that all patients with FLG
mutations have the potential to develop AD, based upon their inherited barrier defect alone, but repeated epicutaneous deposition of allergens, sufficient to provoke TH2-dominant inflammation, might not occur in some IV patients. Second, in mice, unlike humans, development of inflammation might not require the superimposition of additional acquired stressors, because as noted above, mouse skin displays reduced barrier competence in comparison to human skin 63–65
. This inherent difference could also explain why hapten-induced, acute allergic contact dermatitis converts to an AD-like phenotype following repeated challenges in mice, a transition that is not known to occur in humans.
In summary, we show here that FLG deficiency, as occurs in many cases of human AD, suffices to provoke a barrier abnormality, which in turn allows enhanced permeation of water-soluble tracers via the paracellular route. The paracellular defect can be attributed to abnormal extracellular lamellar bilayers, resulting from compromised lamellar body secretion. These structural abnormalities allow enhanced irritant/hapten ingress, resulting in reduced inflammatory thresholds, providing a mechanistic link between FLG deficiency and development of AD. Finally, these studies substantiate and explain the new, ‘outside-to-inside’ paradigm of AD pathogenesis.
- Filaggrin deficiency alone provokes a permeability barrier abnormality (no further acquired stressors are necessary).
- The barrier abnormality localizes to the extracellular spaces of the stratum corneum, where if facilitates bidirectional, paracellular percutaneous transport of water-soluble xenobiotes.
- The paracellular abnormality can be ascribed to impaired secretion of epidermal lamellar bodies.
- The barrier abnormality correlates with reduced inflammatory thresholds to topical irritants and haptens.
- Repeated, low dose hapten applications that fail to elicit inflammation in wild-type mice, provoke inflammation with certain th2 features in filaggrin-deficient mice, worsening the barrier abnormality, thereby completing an ‘outside-inside-(back to) outside’ pathogenic cycle in murine AD.
Short Summary of Clinical Implications
FLG deficiency (in human AD) provokes a paracellular barrier abnormality, caused by abnormal extracellular lamellar bilayers, resulting from compromised lamellar body secretion. These structural abnormalities reduce inflammatory thresholds to irritants/haptens, providing a mechanistic link between FLG deficiency and development of AD, explaining the new, ‘outside-to-inside’ paradigm of AD pathogenesis.