Chronic Proliferative Dermatitis in Mice: NFκB Activation Autoinflammatory Disease

Abstract

Autoinflammatory diseases are a heterogeneous group of congenital diseases characterized by the presence of recurrent inflammation, in the absence of infectious agents, detectable autoantibodies or antigen-specific autoreactive T-cells. SHARPIN deficient mice presents multiorgan chronic inflammation without known autoantibodies or autoreactive T-cells, designated Sharpin(cpdm). Histological studies demonstrated epidermal hyperproliferation, Th-2 inflammation, and keratinocyte apoptosis in this mutant. The mutant mice have decreased behavioral mobility, slower growth, and loss of body weight. Epidermal thickness and mitotic epidermal cells increase along with disease development. K5/K14 expression is distributed through all layers of epidermis, along with K6 expression in interfollicular epidermis, suggesting epidermal hyperproliferation. K1/K10 is only detectable in outer layers of spinosum epidermis, reflecting accelerated keratinocyte migration. Alpha smooth muscle actin is overexpressed in skin blood vessels, which may release the elevated white blood cells to dermis. CD3(+)CD45(+) cells and granulocytes, especially eosinophils and mast cells, aggregate in the mutant skin. TUNEL assay, together with Annexin-V/propidium iodide FACS analysis, confirmed the increase of apoptotic keratinocytes in skin. These data validate and provide new lines of evidence of the proliferation-inflammation-apoptosis triad in Sharpin(cpdm) mice, an NFκB activation autoinflammatory disease.

Figure 1

Chronic hyperproliferative dermatitis with loss of body weight. Longitudinal macroscopic observations at 5 time points (2-, 4-, 6-, 8-, and 10-week) found progressive development of dermatitis in Sharpin^cpdm mutant mice (a)–(e), compared to the representative of wildtype mice at the age of 10 weeks (f). Hair loss and skin redness can be carefully found at forehead in 2-week-old mutant and gradually develop toward the posterior end of body. After the age of 6 weeks, pigmented skin islands formed containing broken hair shafts in skin and inflammatory pigmentation, covered with small, scurfy scales. Skin openings occur at the hairless areas after 6 weeks of age and gradually became deeper and wider until 10-week old. Histologically, increasing of epidermal thickness is consistent with the severity of epidermal hyperproliferation (g)–(m). Keratinocyte mitosis and apoptosis were quantitatively analyzed at each time point (m, n). Compared to wildtypes, the mutant mice present slower growth of body weight at the beginning of 4 weeks of age and lose body weight after 8 weeks of age (p).

Figure 2

Exploratory test showed reduced behavioral activities in Sharpin^cpdm mice. Mutant (a) and wild-type (b) mice were placed in open field with 16 holes on the board. Track distance, track speed, and hole visits were automatically recorded in limited time (c).

Figure 3

Granulocytic inflammation in the skin of Sharpin^cpdm mice. (a, b): Alpha smooth muscle active was overexpressed in the skin blood vessel of mutant mice (a), compared to that of control (b). (c, d): Toluidine blue staining demonstrated increased infiltration of mast cells in the dermis of mutant mice (c). A few of mast cells present in the skin of healthy mice (d). (e, f): Increased T cells (Green, CD3e⁺) migrate into the skin lesion of mutant mice, among which some are helper T cells (double positive, CD3e⁺CD45RB⁺) (e). A few CD3e⁺T cells (Green), but no CD45RB⁺ T cells were detected in the normal control (f). (g)–(l): Gr-1⁺ granulocytes were clustered in the mutant skin (g), compared a few in normal skin for physiological demands (h). Transmission electron microscopy found most granulocytes are eosinophils (i), with moderate mast cells (j) and few neutrophils (k) although neutrophils had huge increase in peripheral blood (l).

Figure 4

Expression changes of keratin and filaggrin in skin. (a, b): K5 expression in the basal layer of the epidermis of 10-week-old wild-type mice (a) but in the whole epidermis, preferentially in the basal layer, of 10-week-old Sharpin^cpdm mice (b). (c, d): K14 expression in the basal layer of the epidermis of 10-week-old wild-type mice (c). K14 was uniformly expressed in the whole epidermis of 10-week-old Sharpin^cpdm mice (d). (e, f): K1 expression locates in suprabasal layer of the epidermis of 10-week-old wild-type mice (e) but limited to the outer layers of the stratum spinosum of 10-week-old Sharpin^cpdm mice (f). (g, h): K10 expression is located at the suprabasal layers of the epidermis and hair follicle of 10-week-old wild-type mice (g) but limited to the outer layers of the stratum spinosum of the epidermis and hair follicle of 10-week-old Sharpin^cpdm mice (h). (i, j): K6 is only expressed in the hair follicle of 10-week-old wild-type mice (i) but widely expressed in both hair follicle and interfollicle epidermis of 10-week-old Sharpin^cpdm mice (j). (k, l): Filaggin expression in the granular layers of the epidermis of 10-week-old normal and mutant mice.

Figure 5

Increased keratinocyte apoptosis in the skin of Sharpin^cpdm mice. (a, b): Numerous apoptosis bodies in the epidermis and hair follicles of 6-week-old Sharpin^cpdm mice (a). No apparent apoptosis bodies were identified in the matched normal skin (b). (c, d): TUNEL immunofluorescence demonstrated that most apoptotic cells were located in the hair follicles, and some in the suprabasal epidermis, of 6-week-old Sharpin^cpdm mice (c). No TUNEL positive cells were found in the epidermis of normal skin (d). TUNEL positive cells also present in the dermis of both mutant and normal skin. (e, f): Annexin-V/propidium iodide FACS analysis confirmed apoptosis in the epidermis of 6-week-old mutants. 15.2% and 18.9% of total epidermal cells of the mutant mice were, respectively, late apoptotic or actually dead cells (e), compared to 1.07% and 7.67% of the wild-type epidermal cells (f).