ULK1 Inhibition as a Targeted Therapeutic Strategy for Psoriasis by Regulating Keratinocytes and Their Crosstalk With Neutrophils


doi: 10.3389/fimmu.2021.714274.


eCollection 2021.

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Xiaonan Qiu et al.


Front Immunol.


.

Abstract

Psoriasis is a common inflammatory skin disease resulting from an interplay of keratinocytes and immune cells. Previous studies have identified an essential role of autophagy in the maintenance of epidermal homeostasis including proliferation and differentiation. However, much less is known about the role of autophagy-related proteins in the cutaneous immune response. Herein, we showed that ULK1, the key autophagic initiator, and its phosphorylation at Ser556 were distinctively decreased in the epidermis from lesional skin of psoriasis patients. Topical application of SBI0206965, a selective ULK1 inhibitor, significantly attenuated epidermal hyperplasia, infiltration of neutrophils, and transcripts of the psoriasis-related markers in imiquimod (IMQ)-induced psoriasiform dermatitis (PsD). In vitro, ULK1 impairment by siRNA and SBI0206965 arrested cell proliferation and promoted apoptosis of keratinocytes but had a marginal effect on the expression of proinflammatory mediators under steady status. Surprisingly, SBI0206965 blocked the production of chemokines and cytokines in keratinocytes stimulated by neutrophils. Of interest, the pro-apoptotic and anti-inflammatory effects of ULK1 inhibition cannot be fully replicated by autophagic inhibitors. Our findings suggest a self-regulatory process by downregulating ULK1 to maintain the immune homeostasis of psoriatic skin via regulating keratinocytes and their crosstalk with neutrophils, possibly through both autophagy-dependent and independent mechanisms. ULK1 might be a potential target for preventing or treating psoriasis.


Keywords:

ULK1 (unc-51 like autophagy activating kinase 1); autophagy; keratinocyte; neutrophil; psoriasis.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures


Figure 1



Figure 1

Decreased expression of ULK1 in psoriatic epidermis. (A) Heatmap of the autophagy-related gene expression determined by microarray (n=4). (B) Expression of ULK1 mRNA (n=5) in epidermis from psoriatic lesions and normal skin from health controls (HC). (C, D) Representative image and quantification of ULK1 and phospho-ULK1 (Ser556) expression by immunohistochemical (IHC) staining in healthy control, lesional skin from patients with eczema or psoriasis (n=8-10). Scale bars, 50 μm. Data are presented as mean± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.


Figure 2



Figure 2

ULK1 inhibitor SBI-0206965 (SBI) ameliorates psoriasiform dermatitis (PsD) induced by imiquimod (IMQ). (A) Representative images and quantification of immunochemical staining of p-ULK1 (ser555) and ULK1 in dorsal skin from mice treated with topical IMQ or vanicream for 7 days (scale bars, 50 μm). (B) Schematic illustration of experimental protocols. (C) p-ULK1 (ser555) expression in dorsal skin from mice treated with 100 µM SBI as shown in (B) on day 0 (scale bars, 25 μm). (D) Manifestations and the severity of skin inflammation measured by PSI score in mice treated as shown in (B). (E) HE staining and histological analysis of epidermis thickness (scale bars, 50 μm). Data are presented as mean± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.


Figure 3



Figure 3

Topical application of SBI therapeutically improves PsD with inhibited infiltration of neutrophils. (A) Schematic illustration of experimental protocols. (B) Manifestations and the severity of skin inflammation measured by PSI score in mice treated as shown in (A). (C) HE staining and histological analysis of epidermis thickness. (D) Ki-67 staining examined by immunohistochemistry staining. (E) Flow cytometry plots showing gating strategy. Absolute number of infiltrating CD45+ cells and neutrophils examined by flow cytometry. (F) mRNA expression of cytokines in the whole ear. 6 animals per group. Scale bars, 50 μm. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.


Figure 4



Figure 4

Inhibition of ULK1 suppresses proliferation and promotes apoptosis of keratinocytes in vitro. (A) Immunoblot of HaCat keratinocytes 24 hours after cocultured with DMSO, 5 µM or 10 µM SBI-0206965 (SBI). (B) Cell cycle analysis of HaCat keratinocytes treated with DMSO or 10 µM SBI for 24 hours. The bar graph shows the percentage of cell population in each phase of cell cycle. (C) Apoptosis of HaCat keratinocytes 24 hours after serum deprivation in the presence of DMSO or 10 µM SBI. (D) mRNA expression of psoriasis-related inflammatory mediators in HaCat keratinocytes cocultured with DMSO or 10 µM SBI for 24 hours. (E) mRNA and protein expression of ULK1 in HaCat keratinocytes transfected with negative control-siRNA(NC-siRNA) and ULK1-siRNA. (F) Cell cycle analysis of HaCat keratinocytes 72 hours after transfection with NC-siRNA or ULK1-siRNA. (G) Apoptosis of transfected HaCat keratinocytes 24 hours after serum deprivation. (H) mRNA expression of psoriasis-related inflammatory mediators in HaCat keratinocytes transfected with NC-siRNA or ULK1-siRNA for 72 hours. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.


Figure 5



Figure 5

Inactivation of ULK1 by SBI suppressed the inflammation in keratinocytes stimulated by neutrophil. (A) mRNA expression of psoriasis-related inflammatory mediators by keratinocytes stimulated with neutrophils isolated from healthy donors (HC) or (B) psoriasis patients in the presence of 10 µM DMSO or SBI for 10 hours. Data are representative of three independent experiments. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.


Figure 6



Figure 6

Autophagy inhibitors fail to fully replicate the effect of ULK1 inhibition on keratinocyte. (A–C) Expression of p62 and LC3 I/II in HaCat keratinocytes 24 hours after incubation with SBI0206965 (SBI) (A) or chloroquine (B) or 3-methyladenine(3-MA) at indicated concentration (C). (D) Cell cycle analysis and (E) apoptosis of keratinocytes after 24 hours with the treatment of 10 µM chloroquine or 5 mM 3-MA. (F) mRNA expression of psoriasis-related inflammatory mediators by keratinocytes cocultured with neutrophils from healthy donors in the presence of 5 mM 3-MA. Data are representative of three independent experiments. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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