Fig 1: TLR4/MyD88/TRAF6/TAK1 and NF-κB signaling mediate the NETs-induced immune responses (A) QRT-PCR for TLR2, TLR4, and Dectin1 with cDNA from cultured keratinocytes stimulated with NETs. Data are expressed as means ± SD (n = 3). Two-way ANOVA. (B) Tissue immunofluorescence analysis of the location and expression of TLR4 in the skin lesions of psoriasis patients and normal controls. Scale bars, 100 μm. (C) The protein expressions of downstream adaptor molecules of TLR4 in NETs-stimulated keratinocytes. (D) The phosphorylation level of phospho-NF-κB p65 and total proteins at indicated time following NETs stimulation in primary keratinocytes. (E) The phosphorylation level of NF-κB p65 in TLR4- or MyD88-silenced keratinocytes with NETs stimulation for 30min. (F) LCN2 expression in TLR4-, MyD88-, or NF-κB p65-silenced keratinocytes with NETs stimulation. (G) The mRNA levels of LCN2 and IL36G in TLR7- or TLR9-silenced keratinocytes followed by NETs stimulation. One-way ANOVA, data are expressed as means ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. All experiments were repeated for at least three times.
Fig 2: NETs activate inflammatory responses in keratinocytes (A) The mRNA expressions of inflammatory mediators in human primary keratinocytes stimulated with PMA-derived NETs or medium from unstimulated neutrophils for 24 h. One-way ANOVA. Data are expressed as means ± SD (human primary keratinocytes were obtained from three donors; peripheral blood samples were obtained from psoriasis patients). (B) The protein level of LCN2, CXCL8, CXCL1, and IL-36? in the supernatant of cultured keratinocytes stimulated with NETs over indicated time. Data are expressed as means ± SD (n = 3). Two-way ANOVA. (C) Short summary of identified NETs-associated proteins that abundant in NETs supernatant that derived from neutrophils of psoriasis patients. (D) The mRNA expressions of LCN2 and IL36G in cultured keratinocytes stimulated with NETs or recombinant proteins over 24 h. One-way ANOVA (adjusted for Dunnett's test). Results represent means ± SD (n = 3). (E) The mRNA levels of LCN2, IL36G, CXCL1, and CXCL8 in keratinocytes stimulated by NETs or the supernatant from PMA-induced neutrophils that had been pre-treated with CI-amidine. One-way ANOVA. Values represent the means ± SD (n = 3). *P < 0.05, **P < 0.01, ****P < 0.0001. All the bars represent the average of three independent experiments. Unstim ctrl, unstimulated control.
Fig 3: TLR4 activation synergizes with IL-36? to induce inflammatory gene expression (A) The mRNA expressions of LCN2 and IL36G in keratinocytes stimulated with recombinant IL-36? (100 ng/ml) or IL-1ß (50 ng/ml) for 24 h. (B,C) The mRNA levels of LCN2, IL36?, and other inflammatory mediators in keratinocytes co-cultured with NETs and anti-IL36R or not. (D) The mRNA expression of LCN2 in keratinocytes stimulated with IL-36? and LPS. (E,F) The mRNA and protein levels of TLR4 and activation of MyD88 in cultured keratinocytes stimulated with IL-36? or IL-1ß for indicated time. (G,H) The mRNA and protein levels of TLR4 and activation of MyD88 in keratinocytes co-cultured with NETs and anti-IL36R or not for indicated time. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA, n = 3 (mean ± SD). All the bars represent the average of three independent experiments.
Fig 4: Inhibition of TLR4 function ameliorates NETs-exacerbated skin inflammation in vivo (A) Representative H&E analysis of the ears on day 7. TLR4 or control siRNA within the cream were applied to the ears of BALB/c mice daily, and NETs were subcutaneously injected back every 48 h. Scale bar = 100 μm. The study involved 3 mice per group and 3 independent experiments. (B) The analysis of skin thickening and inflammatory cell infiltration. Two-way ANOVA, n = 8 (mean ± SD). (C) Ear dermis and epidermis were separated. Western blot analysis of MyD88 and phosphorylated p65 expressions in the epidermis of ears after treatment as in A. (D) The mRNA level of Lcn2 and Il17α in the epidermis of mice subjected to indicated administration as in A. Two-way ANOVA. Values represent the means ± SD (n = 3). (E) Histological sections of mouse ears on day 7. TAK-242 or DMSO within the cream were applied to the inside ear of BALB/c mice daily, and NETs were subcutaneously injected back every 48 h. Scale bar = 100μm, n = 3 per group. (F) Statistical analysis of the epidermal thickness and infiltrated cells in dermis. Two-way ANOVA, n = 8 (mean ± SD). (G) Lcn2 and Il17α expressions in the epidermis of ears. Two-way ANOVA. Values represent the means ± SD (n = 3). (H) Representative H&E staining, infiltrated neutrophils labeled by Ly-6G, and immunofluorescence staining NETs structure in back skin from control IgG or LCN2 mAb treated IMQ mice, n = 5 per group. Scale bar: 100 μm. (I) Statistical analysis of Ly-6G labeled cells in H. Two-tailed Student's t-test, n = 8 (means ± SD). (J) Statistical analysis in back skins of control IgG or LCN2 mAb treated IMQ mice. Two-tailed Student's t-test, n = 3 (means ± SD). Scale bar = 100μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. All the bars represent the average of three independent experiments.
Fig 5: Proposed model of the NETs-TLR4/IL-36R-keratinocyte amplification loop in psoriasis. In psoriasis, neutrophils infiltrating the epidermis undergo NETosis in close proximity to keratinocytes. NETs stimulate keratinocytes to produce high levels of various inflammatory mediators, including LCN2, IL-36?, CXCL8, and CXCL1. Secreted and activated IL-36? induces TLR4 expression. Endogenous neutrophils-derived TLR4 ligands then synergize with IL-36, signaling through MyD88 and NF-kB activation, to induce LCN2 and IL-36? production. In turn, the up-regulated LCN2 modulates NETs formation and neutrophil migration, enhancing and sustaining the inflammatory response. Thus, targeting NETs/TLR4 may provide a novel therapeutic approach for the treatment of psoriasis.
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