Retinoid X receptor ablation in adult mouse keratinocytes generates an atopic dermatitis triggered by thymic stromal lymphopoietin

Abstract

To investigate the role of retinoid X receptors (RXRs) in epidermal homeostasis, we generated RXRalphabeta(ep-/-) somatic mutants in which both RXRalpha and RXRbeta are selectively ablated in epidermal keratinocytes of adult mice. These mice develop a chronic dermatitis mimicking that observed in atopic dermatitis (AD) patients. In addition, they exhibit immunological abnormalities including elevated serum levels of IgE and IgG, associated with blood and tissue eosinophilia, indicating that keratinocyte-selective ablation of RXRs also generates a systemic syndrome similar to that found in AD patients. Furthermore, the profile of increased expression of cytokines and chemokines in skin of keratinocyte-selective RXRalphabeta-ablated mutants was typical of a T helper 2-type inflammation, known to be crucially involved in human AD pathogenesis. Finally, we demonstrate that thymic stromal lymphopoietin, whose expression is rapidly and strongly induced in RXRalphabeta-ablated keratinocytes, plays a key role in initiating the skin and systemic AD-like pathologies.

Fig. 1.

RXRαβ^ep-/- mice develop a chronic skin inflammation. (a-f) Appearance of control (CT) and RXRαβ^ep-/- mutant mice at weeks 2 and 24 as indicated. The white arrowhead in e points to the reddening and thickening of a mutant ear at week 2, to be compared with its normal appearance in a CT mouse (d). Black arrows in c and f point to skin lesions seen at week 24 on the back and the ears, and on the face and the neck, respectively. (g-l) Hematoxylin and eosin-stained sections of ear (g-i) and dorsal skin (j-l) of CT and RXRαβ^ep-/- mice at week 2 and week 12. White arrows point to dermal/epidermal junction. Yellow arrows point to blood vessels. hf, hair follicle; u, utriculi. (Scale bar, 50 μm.)

Fig. 2.

Characterization of inflammatory cell infiltrates in ear skin. Immunohistochemical (IHC) staining was performed on ear sections from control (CT) and RXRαβ^ep-/- mutant (MT) mice at week 8, with antibodies against CD4 (a and b), CD11c (c and d), and MHCII (e and f). Red color corresponds to staining of antibodies, whereas blue corresponds to DAPI staining of nuclei. White arrows point to dermal/epidermal junction. (Scale bar, 50 μm.) (g and h) Luna's staining. Eosinophils (pointed by red arrows in h Inset) display intracytoplasmic red staining. (i and j) Electron microscopic (EM) analyses. Eosinophils with crystal-like rectangular inclusions are pointed by black arrows and shown enlarged in j Inset. (k and l) Toluidin blue-stained sections. In each panel, the black arrow points to one of the mast cells showing intensive blue color. (Scale bars, 50 μmin g, h, k, and l and 2 μmin i and j.)

Fig. 3.

Cytokine and chemokine expression in RXRαβ^ep-/- skin. (a) Quantitative RT-PCR analyses of cytokines and chemokines in ear skin at week 2, week 4 and week 12. Relative expression levels in RXRαβ^ep-/- mutants (MT) compared to controls (CT) (set as 1.0) are shown in Left; IL4 RNA levels of CT (undetectable at week 2-12) and MT are shown in Right. (b) TSLP RNA levels in epidermis of ear skin (ES) and dorsal skin (DS), tongue (To), salivary gland (SG), thymus (Thy), spleen (Sp), lymph node (LN), liver (Li), and lung (Lu) at week 2. ^*, P < 0.05. (c) Immunohistochemical staining of TSLP in ear skin at week 2. Red corresponds to antibody staining, and blue corresponds to DAPI staining of nuclei. White arrows point to dermal-epidermal junction. White arrowheads point to autofluorescence of erythrocytes. (Scale bar, 50 μm.)

Fig. 4.

Systemic immunological abnormalities in RXRαβ^ep-/- mice. (a) IgE and IgG levels in sera of nine controls (CT) and nine mutant (MT) mice at week 12. The average values are indicated by short lines. (b) Lymph node (LN) hyperplasia and splenomegaly in MT mice. Cervical LNs and spleens from a CT and two MT mice (MT1 and MT2) are shown at week 20. The relative weights of cervical LN and spleen to body weights (BW) from CT and MT mice at week 0-20 are presented. (c) Luna's staining of cervical LN, spleen and liver sections (as indicated) at week 12. The white arrow points to one of the many eosinophils (stained red in cytoplasm and blue in nucleus). The black arrowhead points to erythrocytes (stained red in cytoplasm). V, vein. (Scale bar, 25 μm.)

Fig. 5.

RXRαβ^epaf2o mice do not develop a skin inflammation. (a-c) Appearance of control (CT) (a), RXRαβ^ep-/- mutant (b), and RXRαβ^epaf2o mutant (c) at week 5. Close views of the ears are shown in Insets. Black arrows in b and c point to regions with hair loss, and white arrowhead in b Inset points to the red and swollen ear. (d-f) Hematoxylin/eosin staining of ear sections of CT (d), RXRαβ^ep-/- mutant (e), and RXRαβ^epaf2o mutant (f) at week 5. White arrows point to dermal-epidermal junction. hf, hair follicle; u, utriculi. (Scale bar, 50 μm.) (g and h) Comparison of immune cell infiltrate (g) and RNA levels of cytokines and chemokines (h) in ear skin of RXRαβ^ep-/- and RXRαβ^epaf2o mutant mice at week 5. The numbers in g represent the average counting of the corresponding cells from three microscopic fields (objective ×20) of ear sections. ^*, P < 0.05.

Fig. 6.

K14-TSLP transgenic mice develop a chronic skin inflammation. (a-d) Appearance of a 3-week-old (a) and a 7-week-old (b) K14-TSLP transgenic mouse, with closer views of the ears shown in Insets. Photos of skin lesions exhibited by the 7-week-old K14-TSLP transgenic mouse in the neck (c) and on dorsal (d) regions were taken after shaving. White arrowheads point to lesioned skin. (e and f) Hematoxylin and eosin-stained ear sections of 3-week-old control (e) and K14-TSLP transgenic (f) mice. White arrows point to the dermal/epidermal junction. Yellow arrow points to blood vessel. hf, hair follicle. (g and h) Immunohistochemical staining of CD4 on ear sections from 3-week-old control (g) and K14-TSLP transgenic (h) mice. Red color corresponds to the staining of the antibodies, and blue corresponds to the DAPI staining of nuclei. White arrows point to dermal/epidermal junction. (Scale bars, 50 μm.) (i) RNA levels of cytokines and chemokines (as indicated) in ear skin of 3-week-old representative control and K14-TSLP transgenic mice.