We demonstrate that filaggrin deficiency is associated with Th17-dominated skin inflammation, development of eczematous skin lesions with age, and permissiveness to EC sensitization with protein antigen.
Except for matted hair, there were no discernible skin lesions in ft/ft
mice until about 28 weeks of age. By 32 weeks of age, ft/ft
mice developed scaly eczematous skin lesions (). Skin lesions have not been previously described in ft/ft
, possibly because previous reports may not have investigated older mice. Alternatively, as in Nc/Nga mice22
, development of skin lesions in ft/ft
mice may be influenced by environmental factors, particularly microbial skin colonization, which may differ between animal facilities.
Normal appearing skin from 8-week-old ft/ft
mice revealed skin inflammation characterized by hyperkeratosis, acanthosis, dermal infiltration with CD4+
cells and rare neutrophils, with notable absence of eosinophils, and increased expression of mRNA for IL-17, the IL-17 promoting cytokines IL-6 and IL-23, and the IL-17 inducible chemokine CXCL2 (). More severe, but qualitatively similar, skin inflammation was observed in skin lesions that had developed spontaneously in 32-week-old ft/ft
mice (). Keratinocytes, DCs, and macrophages are sources of IL-6 and IL-23, and release these cytokines in response to TLR ligands and IL-123-25
. TLR2 ligands from S. aureus
and other bacteria, which colonize skin are particularly potent at driving IL-23 expression26
. We have found that mechanical skin injury by tape stripping causes rapid upregulation of IL-6 and IL-23 in the skin (MKO and RSG: unpublished observations). There was no significant elevation of IL-6 and IL-23 mRNA expression in the skin of 2-week-old ft/ft
mice (data not shown) suggesting that lack of filaggrin by itself does not drive increased expression of these cytokines. The trigger for this increase could be microbial antigens, absorbed via a disrupted skin barrier, and/or a mechanically damaged skin. Elevated IL-17 expression may explain the neutrophil infiltration in ft/ft
skin, as IL-17 induces expression of neutrophil chemotactic chemokines in epithelial cells, including CXCL227
, which was upregulated in ft/ft
skin. In addition to T cells, neutrophils are a source of IL-1719
, and may contribute to increased IL-17 expression in ft/ft
skin. IL-6 drives keratinocyte proliferation28
, thus increased IL-6 expression in the skin may underlie epidermal thickening in ft/ft mice.
The skin of 8-week-old ft/ft
mice exhibited no detectable evidence of Th2-driven inflammation. There was no significant infiltration with eosinophils or upregulation of Th2 cytokine mRNA expression (). There was also no evidence of Th1-driven inflammation, as there was no increase in IFN-γ mRNA expression. Lack of evidence of Th2 inflammation in the skin of ft/ft
mice contrasts with elevation of their serum IgE and IgG1 levels, an indicator of a systemic Th2 response. High levels of IL-17 in the skin of ft/ft
mice may have inhibited local expression of Th2 cytokines and subsequent recruitment of eosinophils29
. Skin lesions in 32-week-old ft/ft
mice exhibited qualitatively similar, but more pronounced, changes in histology and cytokine expression, compared to skin from 8-week-old ft/ft
mice () The exception was IL-4 mRNA levels, which were elevated in lesional skin of 32-week-old, but not 8-week-old mice, consistent with an ongoing Th2 response to cutaneously absorbed antigens.
The skin of ft/ft
mice was permissive to EC sensitization of nonstripped skin with OVA, a protein antigen, normally excluded from penetrating the skin. Application of OVA to shaved, but non-tape stripped, skin elicited allergic skin inflammation and OVA specific systemic humoral and cellular immune responses in ft/ft
mice, but not C57BL6 or BALB/c mice ( and ). In previous studies, two additional strains, 129Sv and hr/hr hairless mice, failed to respond to OVA application to shaved skin (RSG: unpublished observations). Skin inflammation in EC sensitized ft/ft
mice was characterized by a significant increase in epidermal thickness, dermal infiltration with CD4+
cells and expression of IL-4, IL-17 and IFN-γ, but not IL-13 or IL-5 mRNA. IL-13 plays an important in driving eotaxin expression in the skin30
. IL-5 is important not only for eosinophil mobilization, but also for their survival in tissues31
. Elevated IL-17 levels in ft/ft
skin before, and more so after EC sensitization, may have inhibited of IL-13 and IL-5 expression, resulting in lack of eosinophil infiltration. Genetic background could have contributed to lack of IL-5 and IL-13 mRNA expression in EC sensitized skin of ft/ft
EC sensitization of tape stripped skin of WT mice (BALB/c, C57BL6, 129Sv and BWF1) results in allergic skin inflammation and in OVA specific immune responses16, 32, 33
. Since tape stripping disrupts skin barrier and allows antigen entry34
, disrupted skin barrier function in ft/ft
mice likely underlies their permissiveness to EC sensitization of nonstripped skin by protein antigen. This permissiveness did not reflect a generalized enhanced immune response, because the humoral and immune responses of ft/ft
mice to i.p.
immunization with OVA were comparable or lower than those of C57BL6 and BALB/c mice (). This is consistent with the observation that FLG
is expressed exclusively in mouse skin, tongue, esophagus, and forestomach35
. With their eczematous skin lesions that developed with age and expressed IL-4 mRNA, elevated serum IgE and IgG1 levels and permissiveness to EC sensitization of nonstripped skin with protein antigen, ft/ft
mice share features with patients with extrinsic AD. However, while IL-17 expression is prominent in the skin of ft/ft mice and in psoriatic skin lesions, it is less prominent in AD skin lesions. There are two limitations to the interpretation of our results. Although mutation in FLG
likely underlies the skin abnormalities in ft/ft
mice, these mice are double homozygous of flaky tail
. Therefore, we cannot rule out a role for the ma
mutation in the skin inflammation and susceptibility to EC sensitization of nonstripped skin of ft/ft
mice. The other limitation is that the ft/ft
mice we studied, are not on a homogeneous background. Nevertheless, the skin inflammation and susceptibility to EC sensitization of nonstripped skin we observed in these mice was not observed in four other strains we have studied to date. Thus, it is unlikely that genes other than FLG
, or a closely linked gene, are implicated in the susceptibility of ft/ft
mice to develop skin inflammation to protein antigens normally excluded by the skin barrier and to develop eczema.
While this manuscript was being reviewed, the mutation in FLG
mice was identified to be a 1-bp deletion (5303delA), and ft/ft
mice bred on C57BL6 background and lacking the ma mutation were demonstrated to be permissive to sensitization by cutaneous application of antigen, in agreement with our findings36
Filaggrin deficiency predisposes to skin inflammation, development of eczematous lesions and sensitization upon contact with protein antigens normally excluded by the skin barrier.