Synergistic effects of LCN2 and TWEAK on the progression of psoriasis

Abstract

Lipocalin 2 (LCN2) and the TWEAK/Fn14 signaling pathways are pivotal in psoriasis, influencing epidermal development, inflammatory cell chemotaxis, and inflammatory factor release. Despite their significant roles, the intricate relationship between LCN2 and TWEAK/Fn14 pathways remains unclear. Our study revealed the correlation between the expression of TWEAK, LCN2, and Fn14 in psoriatic lesions. We found that TWEAK is expressed by keratinocytes and macrophages, while LCN2 is expressed by keratinocytes and neutrophils. Surface plasmon resonance experiments demonstrated binding between LCN2 and Fn14, which was further validated by co-immunoprecipitation and cellular co-localization via immunofluorescence. In vitro, LCN2 promoted macrophage differentiation and TWEAK secretion, enhanced TWEAK and Fn14 expression in keratinocytes, and activated the MAPK signaling pathway. TWEAK upregulated LCN2 expression in neutrophils but not in keratinocytes. Bulk RNA-seq analysis revealed a synergistic effect of LCN2 and TWEAK in promoting inflammatory cytokine expression in keratinocytes, with enhanced MAPK pathway activation in the presence of M5 cytokines. Lcn2 knockout reduced Fn14 expression in skin lesions and serum TWEAK levels of imiquimod-induced murine psoriasis model, while Fn14 knockout attenuated the epidermal hyperplasia-promoting effects of TWEAK and LCN2. Overexpression of Fn14 in keratinocytes led to higher TWEAK expression upon LCN2 stimulation, suggesting a self-reinforcing loop among TWEAK, LCN2, and Fn14. We propose that LCN2 synergizes with TWEAK through Fn14 to drive psoriasis pathogenesis.

Keywords: Fn14; Lipocalin 2; Psoriasis; TWEAK; synergistic.

Fig. 1

TWEAK, Fn14, and LCN2 are overexpressed in skin lesions of psoriasis patients and IMQ induced psoriatic mice model. A Single cell data from psoriasis was used for correlation analysis between TWEAK, Fn14 and LCN2. B Immunostaining of LCN2, TWEAK and Fn14 in the skin of healthy control (HC) and patient with psoriasis (Brown). Normal control skin samples = 4, patient skin samples = 4. Nuclei were stained with hematoxylin (Bar = 200 μm). C Immunostaining of LCN2, TWEAK and Fn14 were performed on wild-type mouse skin paraffin sections. Stained epidermal region areas were quantitated by ImageJ software. The relationship between the IOD of TWEAK (or Fn14) and LCN2 staining was assessed by Spearman’s rank correlation. Number of control mice = 3, Number of IMQ induced WT mice = 4. Representative images are shown (Bar = 200 μm). D Volcano Plot showed the RNA-seq analysis of wild-type control mice and wild-type imiquimod model mice. E RNA-seq analysis of wild-type mice and Lcn2^–/– mice treated with imiquimod. F Immunostaining of TWEAK, Fn14 and Ly6G were performed on Lcn2 knockout mice skin paraffin sections. The relationship between the IOD of TWEAK (or Fn14) and number of Ly6G positive cell staining was assessed by Spearman’s rank correlation. Number of control mice = 3, Number of IMQ induced WT mice = 4, Number of IMQ induced Lcn2^–/– mice = 4. Representative images are shown (Bar = 200 μm)

Fig. 2

Expression localization of TWEAK and LCN2 and the interaction between LCN2 and Fn14. A Tissue mIHC results showing the cellular localization of TWEAK expression and its relationship with CD68-labeled macrophages; B tissue mIHC results showing the cellular localization of LCN2 expression and its relationship with CD66b-labeled neutrophils (Bar = 200 μm); C cellular immunofluorescence results showing the colocalization of LCN2 and Fn14; D the binding affinity of LCN2 conjugate to the Fn14 molecule was determined by SPR analysis. E Co-IP experiment detecting the interaction between LCN2 and Fn14 after stimulating Hacat cells cultured in vitro with LCN2

Fig. 3

LCN2 induces the differentiation and secretion of TWEAK in macrophages. A, B Tnfsf12, Tnfrsf12a, and Il6 were detected by RT-qPCR after treating with 0, 10, 50 ng/mL LCN2. 24p3r, Mc4r, inos, and Il10 mRNA in J774A.1 cell were detected by RT-qPCR after treated with 50 ng/mL LCN2. C The intracellular levels of Fn14, TWEAK, p-NFκB P65, p-TRAF2, and TRAF2 proteins were detected by Western blotting after stimulation of J774A.1 cell with LCN2 at concentrations of 0, 10, 50 ng/mL, respectively. D, E TWEAK and CD163 was detected by immunofluorescence in J774A.1 cell after LCN2 treatment. Bar = 100 μm. F Elisa was used to detect the concentration of TWEAK in cell supernatants 24 h after LCN2 stimulation of J774A.1. Data from immunofluorescence assays and Elisa assays are representative of three or more independent experiments are presented as the means ± SEM of triplicate wells. qPCR data are presented as the means ± SEM for triplicate wells from three or more independent experiments. ANOVA was used for comparison between groups: *P < 0.05, **P < 0.01, and ***P < 0.001. ns not significant

Fig. 4

LCN2 regulates the expression of TWEAK and Fn14 and MAPK pathway in keratinocytes. A Immunofluorescence was used to determine TWEAK level after treatment with LCN2 in HFKs. Bar = 50 μm. B Western blotting was used to detect the expression of p-ERK1/2, p-JNK, p-p38, p-TRAF2 and Fn14 in primary keratinocytes after treating the cells with 0, 100, 250 and 500 ng/mL LCN2, respectively. C After the addition of 500 ng/mL LCN2 stimulation, keratinocytes were collected at 0, 0.5, 1, 2, 6, 12, 24 h for WB assay to detect p-ERK1/2, p-JNK, p-p38, p-TRAF2 and Fn14 expression over time. Data from immunofluorescence assays and immunoblot assays are representative of three or more independent experiments are presented as the means ± SEM of triplicate wells

Fig. 5

TWEAK promotes neutrophil expression of LCN2 but enhances inflammation in keratinocytes by inducing MMP9 expression. A Relative mRNA expression level of Lcn2, Tnfrsf12a, Tgfb1, Il6, Tnfa, Il1b, Krt5, Krt10, Krt17, 24p3r, Mc4r in mouse skin treated with TWEAK was detected by RT-qPCR. B Immunostaining of LCN2 and Ly6G were performed on paraffin sections of IMQ mice model with or without dorsally topical application of rmTWEAK (20 μg/mL, prepared in PBS). Unpaired two-tailed t test. C Immunofluorescence assays were used to detect LCN2 expression in neutrophils after TWEAK stimulation. Bar = 20 μm. D RT-qPCR was conducted to detect Lcn2 mRNA level in neutrophils treated with TWEAK. E LCN2 levels in neutrophil supernatants after TWEAK stimulation for 24 h were measured by ELISA. F Proteomic analysis was used to further investigate the protein changes and possible pathways following TWEAK action on keratinocytes. G After treating with TWEAK on primary keratinocyte with or without M5 cytokines for 48 h, LCN2 and 24P3R protein levels in keratinocytes were detected by Western blotting. H After the TWEAK stimulus, proteins such as MMP9 and TRAF2 were detected by Western blotting. Data from immunofluorescence assays and ELISA assay are representative of three or more independent experiments are presented as the means ± SEM of triplicate wells. ANOVA was used for comparison between groups: *P < 0.05, **P < 0.01, and ***P < 0.001. ns not significant

Fig. 6

The synergistic interaction between LCN2 and TWEAK in keratinocytes. A Heatmap showing differential expressed genes after co-stimulating with TWEAK and LCN2 in keratinocytes. B Relative expression levels of IL6, TNFa, IL17A, CCL5, CXCL5, CXCL10 and 24P3R were detected from HFKs using RT-qPCR. C, D Western blot was used to detect p-ERK1/2, p-JNK, p-p38, p-TRAF2, 24P3R and Fn14 protein level in keratinocytes after stimulation with LCN2, TWEAK, or both. One-way ANOVA was used for comparison between groups: ns (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 7

Lcn2^–/– mice attenuate the pro-proliferative and inflammatory effects of IMQ on the epidermis. A After being treated with imiquimod and LCN2, the expression levels of Fn14, p-ERK1/2 and in the skin of wild-type mice were analyzed by Western blotting (n = 3 per group). B Relative expression levels of Tnfsf12(Tweak), Tnfrsf12a(Fn14), Il6, Tnfa, Il1b, Tgfb1, Krt5, Krt10, and Krt17 from mice skin were detected using RT-qPCR. C, D Photographs of skin lesions in mice and the mouse psoriasis severity score difference between wild-type imiquimod mice and Lcn2^–/– imiquimod model mice. Body weight changes in mice modeled with imiquimod for six consecutive days. E Mouse serum levels of TWEAK were assayed by ELISA. F Western blotting was used to detect KRT1, KRT5, KRT6, KRT10, KRT14, KRT16, KRT17, Loricrin, Fn14, CXCL10, LCN2, p-ERK1/2, p-JNK, p-p38 in wild-type and Lcn2^–/– mice after treated with imiquimod (n = 3 per group). G Relative mRNA expression levels of Lcn2, Tnfsf12, Tnfrsf12a, Il6, Tnfa, Tgfb1, Krt5, Krt10, Krt17, 24p3r, Mc4r, Il1b were detected by RT-qPCR (n = 3–5 per group). H Immunostaining of KRT5 and KRT14 were performed on paraffin sections. Data are shown as mean ± SEM (n = 3–5 per group). ANOVA was used for comparison between groups: ns (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 8

LCN2 acts on the downstream MAPK signaling pathway by binding to Fn14, and the deletion of Fn14 can counteract the effect of TWEAK or LCN2. A KRT6, KRT16, LCN2 were detected by WB in the skin of wild-type mice as well as in the Fn14^–/– mouse imiquimod model. (n = 3 per group). B Immunohistochemistry was used to detect the expression of KRT5 and KRT14 in the skin of wild-type mice and Fn14^–/– mice (Brown). Nuclei were stained with hematoxylin (Bar = 200 μm). C WB detected p-ERK1/2, p-JNK and p-p38 expression in wild-type mice and Fn14^–/– mouse. (n = 3 per group). D Tnfsf12, Tnfrsf12a, Lcn2, Il1b, Il6, Krt5, Krt16, and Krt17 mRNA expression levels were detected by using RT-qPCR methods. E Immunohistochemistry was used to detect the levels of LCN2 and Ly6G in the epidermis of Fn14^–/– mouse imiquimod model after administration of TWEAK. F After treated by LCN2 or TWEAK, Western blot was used to detect the expression of KRT5, KRT6, KRT14, and KRT16 in the Fn14^–/– mice skin tissue. (n = 3 per group) Data were pooled from three independent experiments. Data are shown as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. SPR surface plasmon resonance