1. Increased Lipocalin-2 Contributes to the Pathogenesis of Psoriasis by Modulating Neutrophil Chemotaxis and Cytokine Secretion 
Shuai Shao, Tianyu Cao, Liang Jin, Bing Li, Hui Fang, Jieyu Zhang, Yuan Zhang, Jinhong Hu, Gang Wang 
Journal of Investigative Dermatology 
Volume 136, Issue 7, Pages (July 2016)
DOI: /j.jid
Copyright © 2016 The Authors Terms and Conditions

2. Figure 1
Lcn2 expression is increased in the lesional skin of psoriatic patients and IMQ-treated mice. (a) Lcn2 expression was evaluated using real-time PCR and (b) western blot in the epidermis and the dermis. The skin samples were treated with 2.5 mg/ml dispase for 8 hours at 4°C. (c) Representative images of immunofluorescence of Lcn2 in normal and psoriatic skin. (d) Lcn2 expression levels in the epidermis and the dermis of IMQ-induced mice were determined by real-time PCR and (e) western blot. Skin samples were from a control (○) or IMQ-induced mice (●). (f) Representative immunohistochemical staining of Lcn2 in the back skin of controls and IMQ-treated mice. Scale bar = 100 μm. Values are presented as mean ± standard deviation. ***P < IMQ, imiquimod; Lcn2, lipocalin-2.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

3. Figure 2
Lcn2 mAb/rmLcn2 alleviates/aggravates epidermal hyperplasia and inflammation in IMQ-induced psoriasis-like mice. (a) Mice were intraperitoneally injected with an anti-Lcn2 mAb, isotype control antibody, recombinant Lcn2 protein, or vehicle, respectively, on the first day and every 48 hours thereafter. (b) Phenotype (upper panel) and hematoxylin and eosin staining (lower panel) of IMQ-treated mice in different groups on day 7. Images are representative of three individual mice per group. (c) Epidermal thickness and infiltrated cells in the dermis. (d) The mRNA expressions of important cytokines and (e) immune cell markers. (f) Neutrophil infiltration was determined by evaluating the intensity of Ly6G staining. Scale bar = 100 μm. *P < 0.05, **P < 0.01, ***P < Each experiment was repeated at least 3 times. IMQ, imiquimod; Lcn2, lipocalin-2; mAb, monoclonal antibody.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

4. Figure 3
Lcn2 stimulates the production of critical cytokines by neutrophils. (a) The expression levels of Lcn2 receptors in neutrophils from psoriatic patients and healthy controls. (b) Immunofluorescence analysis of 24p3R on the peripheral neutrophils of psoriatic patients. Scale bar = 10 μm. (c) The colocation of 24p3R and CD15 in the psoriatic dermis. Scale bar = 100 μm. (d) Neutrophils from psoriatic patients were treated with 500 nM of rmLcn2 for the indicated time, followed by real-time PCR analysis of IL-1α, IL-8, IL-6, and TNF-α mRNA expression. (e) Protein levels of IL-1α, IL-8, IL-6, and TNF-α in the supernatant of rmLcn2-stimulated neutrophils. Values are presented as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < Lcn2, lipocalin-2; TNF-α, tumor necrosis factor-α.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

5. Figure 4
The Erk1/2 and p38 MAPK pathways are activated by Lcn2 in neutrophils from psoriatic patients. (a) The phosphorylated p38 MAPK and Erk1/2 levels in neutrophils treated by 500 nM of rmLcn2 were determined by western blot. (b) The nuclear translocation of Erk1/2 and p38 in rmLcn2-stimulated neutrophils. Scale bar = 10 μm. (c) ELISA was employed to assess the inhibiting effects of PD98059 and SB on cytokine secretion from rmLcn2-stimulated neutrophils. (d) Differentiated HL-60 cells were transfected with 24p3R siRNA. (e) The phosphorylated p38 MAPK and Erk1/2 levels and protein levels of cytokines (f) in dHL60 cells stimulated by 500 nM of rmLcn2 for 60 minutes. Similar results were obtained from three separate experiments. *P < 0.05, **P < 0.01, ***P < dHL-60, differentiated HL-60; Erk, extracellular signal-regulated kinase; Lcn2, lipocalin-2; MAPK, mitogen-activated protein kinase; siRNA, small interfering RNA.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

6. Figure 5
Lipocalin-2 (Lcn2) induces the migration of human neutrophils in vitro. (a) A transwell experiment was used to determine the migration of neutrophils. A total of 2 ×105 peripheral neutrophils from psoriatic patients were allowed to migrate for 45 minutes toward different concentrations of rmLcn2. N-formylmethionyl-leucyl-phenyl-alanine (10 nM) was a positive control. The chemotaxis effect was determined by crystal violet staining for the lower transwell membrane (left panel) and counting the number of cells in the lower chamber (right panel). (b) Migration of neutrophils from psoriatic patients was assessed after preincubation with PD98059 or SB for 30 minutes. (c) Migration of neutrophils from psoriatic patients was assessed after treatment with various concentrations of recombinant lipocalin-2 (rmLcn2). N = 8. *P < 0.05, **P < 0.01.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

7. Figure 6
Lipocalin-2 (Lcn2) is mainly produced by activated keratinocyte and infiltrated neutrophils in psoriasis. (a, b) Keratinocytes were stimulated with of IL-17A (20 ng/ml), IL-22 (20 ng/ml), TNF-α (50 ng/ml) or H2O2 (800 nM), and expression levels of Lcn2 were evaluated by western blot (a) and ELISA (b). (c) Colocalization of Lcn2 and CD15+ neutrophils in psoriatic dermis and Munro’s macroabscesses was determined by immunofluorescence. Scale bar = 100 μm. (d) The expression levels of Lcn2 mRNA in circulating neutrophils from psoriatic patients and control subjects. ***P < (e) Immunofluorescence analysis of Lcn2 in neutrophils from psoriatic patients. Scale bar = 10 μm.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions