Genomic control of inflammation in experimental atopic dermatitis

Abstract

Atopic Dermatitis (AD) or eczema, a recurrent allergic inflammation of the skin, afflicts 10-20% of children and 5% adults of all racial and ethnic groups globally. We report a new topical treatment of AD by a Nuclear Transport Checkpoint Inhibitor (NTCI), which targets two nuclear transport shuttles, importin α5 and importin β1. In the preclinical model of AD, induced by the active vitamin D₃ analog MC903 (calcipotriol), NTCI suppressed the expression of keratinocyte-derived cytokine, Thymic Stromal Lymphopoietin (TSLP), the key gene in AD development. Moreover, the genes encoding mediators of T_H2 response, IL-4 and its receptor IL-4Rα were also silenced together with the genes encoding cytokines IL-1β, IL-6, IL-13, IL-23α, IL-33, IFN-γ, GM-CSF, VEGF A, the chemokines RANTES and IL-8, and intracellular signal transducers COX-2 and iNOS. Consequently, NTCI suppressed skin infiltration by inflammatory cells (eosinophils, macrophages, and CD4 + T lymphocytes), and reduced MC903-evoked proliferation of Ki-67-positive cells. Thus, we highlight the mechanism of action and the potential utility of topical NTCI for treatment of AD undergoing Phase 1/2 clinical trial (AMTX-100 CF, NCT04313400).

Figure 1

Graphic Depiction of Experimental Atopic Dermatitis and Treatment Protocol. Atopic dermatitis (AD)-like phenotype was induced in 8-week-old C57BL/6 female mice by topical application of MC903 (calcipotriol), an analog of vitamin D3. On day 1, an initial bolus dose of 6 nmoles of MC903 was applied topically to the right ear (3 nmoles on each side of the ear). On day 3, dosing once a day (Q.D.) with 2 nmoles of MC903 (1 nmole on each side of the right ear) was begun and continued for 3 weeks. On day 20, a 9-day twice-daily (B.I.D.) topical treatment with 66 nmoles NTCI (cSN50.1 peptide, 33 nmoles on each side of the right ear), or an equal volume of NTCI vehicle (10 µl saline on each side of the right ear) was initiated. Mice were weighed and ear thickness was measured every 2–4 days. Ear samples were collected on day 29 for gene expression and Immunohistochemistry (IHC) analyses. Mice in the Mock Control group were only subjected to the measurements of body weight and the ear thickness.

Figure 2

NTCI treatment reduces signs of MC903-induced Atopic Dermatitis (AD)-like phenotype manifested by swelling, redness, and scaling. Topical NTCI treatment accelerates healing in an experimental model of AD-like phenotype. Cardinal signs of inflamed skin, swelling (A), and redness and scaling (C) were significantly reduced by topical application of cSN50.1 peptide. (A) Ear thickness data is displayed as mean ± S.E.M. (n = 3 for Mock Control and n = 10 for all other conditions). Statistical significance of the difference between ear thickness in NTCI-treated mice (MC903 + cSN50.1) and untreated (MC903 Untreated) or saline-treated (MC903 + saline) groups was determined by two-way ANOVA using corrected Holm-Sidak test for multiple comparison, *p < 0.05, **p < 0.005, ***p < 0.0005. (B) Body weight was not significantly affected by MC903 challenge, NTCI, or by saline treatment albeit body weight gain was arrested in these groups. (C) Representative pictures of mice from the Mock Control group taken at the end of experiment and from the cSN50.1-treated group immediately before (MC903 AD Induction) and after NTCI treatment (MC903 + cSN50.1).

Figure 3

The expression of genes encoding the mediators of skin inflammation is suppressed by NTCI. The expression of genes encoding the mediators of allergic inflammation in the ear samples (see Methods and Fig. 1 for a depiction of AD-like phenotype induction and treatment protocol) was determined using a real-time quantitative reverse transcription PCR (qRT PCR). The relative levels of expression were established using Livak’s methods (2^−∆∆Ct) with the 18S gene as a reference and the Mock Control (unchallenged ear) group as the calibrator. Data is presented as a mean + S.E.M. (n = 3 for Mock Control, n = 5 for all other conditions). A statistical analysis was performed using an ordinary one-way ANOVA with an uncorrected Fisher’s LSD test for a multiple comparison, *p < 0.05, **p < 0.005, ***p < 0.0005. The significance levels displayed over the bars of MC903 + Saline and MC903 + cSN50.1 columns represent the statistical difference of each compared to MC903 Untreated samples.

Figure 4

The attenuation of MC903-induced skin inflammation in the murine pinna by NTCI (cSN50.1 peptide) treatment. (A) Exposure to MC903 without treatment or treated with a saline control leads to cellular infiltration comprising predominately macrophages (Iba-1) and eosinophiles (major basic protein or MBP) combined with an influx of CD4 + T cells (CD4), leading to the thickening of the pinna as compared to the mock control. Moreover, the significant immunodetection of Ki67-positive cells indicated increased cellular proliferation in the basal cell layers of the epidermis in response to MC903. Treatment with NTCI (cSN50.1 peptide) reduced the cellular mediators of the inflammatory response. NTCI reduced skin edema (see also Fig. 2A). Shown are representative images of pinna samples with immunohistochemistry for key inflammatory cell markers (× 20 magnification). (B) A semiquantitative analysis of immunohistochemical (IHC) staining presented in panel A. Scoring was conducted according to following formulas: IHC scoring (all inflammatory cell markers): 0—No or almost undetectable immunoreactivity (IR); 1—Scattered and rare IR in the dermis; 2—Multifocal and barely coalescing IR in the dermis; 3—Diffuse IR in the dermis. IHC scoring for Ki67 in epidermis: 0—None; 1—Single cell layer IR at basal zone of epidermis; 2—IR 2—5 layers thick within the basal zone of epidermis; 3—IR > 5 cell layers thick within the basal zone of epidermis. The data is presented as a mean + S.E.M. (n = 5). The statistical analysis was performed using ordinary one-way ANOVA with an uncorrected Fisher’s LSD test for a multiple comparison, *p < 0.05, **p < 0.005.

Figure 5

NTCI treatment reduces PMA-Induced ear inflammation. A single dose of Phorbol Myristate Acetate (PMA, 2 nmoles in 10 µl EtOH on each side) was administered topically to the right ear of 8-week-old female C57Bl/6 mice. Topical treatment with NTCI, cSN50.1 peptide (16.7 nmoles in 10 µl EtOH on each side), or vehicle (10 µl EtOH on each side) was administered 30 min before and 3, 6, and 8 h. after PMA challenge. A control group of mice was treated topically with vehicle only (10 µl EtOH) following the same treatment schedule. (A) Ear thickness was measured at 30 min before (0 h) and 3, 6, 8 and 24 h. after PMA challenge. Measurements are represented as mean ± S.E.M. (n = 5). Statistical significance of the difference between ear thickness in NTCI-treated mice (PMA + cSN50.1) and vehicle treated (PMA + Vehicle) or vehicle control (Vehicle) groups was determined by two-way ANOVA using corrected Holm-Sidak test for multiple comparison, ***p < 0.0005. (B) Ear images show increased swelling and redness induced by PMA (PMA + Vehicle). H & E staining of paraffin-embedded ear punch biopsies, collected 8 h. after PMA challenge, show increased swelling. Both signs of inflammation (redness and swelling) are reduced by NTCI treatment (PMA + cSN50.1). Displayed are representative images (× 20 magnification) of pinna sections.

Figure 6

Mechanism of NTCI action in AD through targeted inhibition of importins α5 and β1 comprising the nuclear transport checkpoint. Proinflammatory Stress-Responsive Transcription Factors, which are activated by allergic, autoimmune, and microbial inducers of inflammation are ferried to the cell’s nucleus through an Importin α5-mediated pathway. Metabolic Transcription Factors, which are activated by overfeeding or inborn errors of metabolism are shuttled to the nucleus by Importin β1. NTCI stops binding of Transcription Factors to Importin α5 and Importin β1 thereby preventing the activation of the genes encoding mediators of Atopic Dermatitis as well as genes encoding metabolic intermediates. The latter underlie the link between AD and Metabolic Syndrome (see text and Suppl. Fig. S1 for expanded view of proinflammatory signaling to the nucleus comparing NTCI action with currently used drugs in AD). NPC, Nuclear Pore Complex.