Neutrophil Extracellular Traps Promote Inflammatory Responses in Psoriasis via Activating Epidermal TLR4/IL-36R Crosstalk

Epidermal infiltration of neutrophils is a hallmark of psoriasis, where their activation leads to release of neutrophil extracellular traps (NETs). The contribution of NETs to psoriasis pathogenesis has been unclear, but here we demonstrate that NETs drive inflammatory responses in skin through activation of epidermal TLR4/IL-36R crosstalk. This activation is dependent upon NETs formation and integrity, as targeting NETs with DNase I or CI-amidine in vivo improves disease in the imiquimod (IMQ)-induced psoriasis-like mouse model, decreasing IL-17A, lipocalin2 (LCN2), and IL-36G expression. Proinflammatory activity of NETs, and LCN2 induction, is dependent upon activation of TLR4/IL-36R crosstalk and MyD88/nuclear factor-kappa B (NF-κB) down-stream signaling, but independent of TLR7 or TLR9. Notably, both TLR4 inhibition and LCN2 neutralization alleviate psoriasis-like inflammation and NETs formation in both the IMQ model and K14-VEGF transgenic mice. In summary, these results outline the mechanisms for the proinflammatory activity of NETs in skin and identify NETs/TLR4 as novel therapeutic targets in psoriasis.

For immunohistochemistry of mouse samples, 4μm sections of paraffin embedded skins were blocked at room temperature with 5% goat serum in PBS for 30 min, and incubated with rat anti-Ly-6G (1:500, Biolegend) at 4°C overnight, followed by HRP labeled goat anti-rat antibodies (1:100, CWBIO) for 30 min at room temperature. DAB revelation (Gene tech) was used for detecting the biotinylated antibodies.

H&E staining and analysis
Normal and lesional skin from donors and mice were fixed in 4% paraformaldehyde, embedded in paraffin, sliced, and stained with hematoxylin and eosin for histological analysis. Epidermal thickness represents the maximal epidermal thickness as measured from the junction of the stratum corneum to the deepest portion of the rete ridge (in humans) or inter-follicular dermis (in mice). Number of the infiltrated inflammatory cells in dermis was counted in eight fields (40μm×40μm) per section, which was manually counted by two independent blinded observers.
Nuclear DNA was detected by incubating cells with Hoechst 33342 (1:1000, Sigma) for 20 min at room temperature. Confocal images were acquired using Olympus Fluoview 1000 microscope with a PlanApo N (×40 with and without a 2.5 digital zoom).

Flow cytometry
To evaluate the neutrophil spontaneous death, peripheral neutrophils were collected and isolated from psoriasis patients and healthy controls by Miltenyi column.
Human neutrophils were then cultured for the 3h, 12h, and 24h. Cells were then harvested, washed twice with ice-cold PBS, and stained with human annexin V (FITC labeled) first and then PI following a protocol provided by the manufacturer (Annexin V-FITC/PI Detection Kit; 7 Seabiotech). FACS was performed using a FACS Canto flow cytometer (BD). In this FACS analysis, cell debris were eliminated by appropriate gating on forward and side scatter. Neutrophil spontaneous death was calculated as the percentage of PI+ cells at each time point.

Small interfering RNA
The small interfering RNA (siRNA) for human TLR4, TLR7, TLR9, MyD88, NF-κB, and control siRNA were synthesized from Ribobio. Keratinocytes were processed with siRNA (5nM) and Lipofectamine 3000 (Invitrogen) following the instructions inside the siRNA kit. After 48h for gene silencing, NETs at indicated concentration were added to each well for stimulation.

Western blot analysis
Western blot assays were performed using whole cell lysates from primary keratinocytes. In short, lysates prepared from cells in a lysis buffer containing a protease inhibitor cocktail (Roche) and a phosphatase inhibitor cocktail (Thermo Fisher Scientific) were separated with SDS-polyacrylamide gel electrophoresis gels. The following antibodies were used: Membranes were incubated overnight using the (1:1000, Abcam), and β-actin (1:5000, CWBIO) as an internal control.

ELISA
LCN2, CXCL1, CXCL8, and IL-36γ levels in the cell-free supernatants from nonstimulated or NETs-stimulated keratinocytes were measured using ELISA assay kits (Neobioscience) following the instructions inside the ELISA kits. Supernatants were stored at -20°C until use for ELISA.

Trypan blue
To determine cell viability of peripheral neutrophils isolated from psoriasis patients and healthy controls, we performed trypan blue exclusion assays according to the instructions (Thermo Fisher Scientific). Experiments were repeated for three times.