Neutrophil extracellular traps are induced in a psoriasis model of interleukin-36 receptor antagonist-deficient mice

Loss-of-function mutations in the interleukin (IL)-36 gene IL36RN are associated with psoriasis. The importance of neutrophil extracellular traps (NETs), web-like structures composed of neutrophil DNA, in the pathogenesis of psoriasis has been unclear. Here, we aimed to clarify the role of NET signaling in the deficiency of IL36 receptor antagonist (DITRA). We evaluated the severity of psoriasis-like lesions induced by imiquimod cream treatment in Il36rn−/− mice. The mRNA levels of psoriasis-related cytokines were measured via real-time reverse transcription polymerase chain reaction, and the effects of Cl-amidine, a peptidyl arginine deiminase 4 (PAD4) inhibitor, on psoriasis-like lesions were evaluated. PAD4 is a histone-modifying enzyme that is involved in NET formation. Psoriasis area and severity index scores, epidermal thickness, and infiltrated neutrophil counts were significantly increased in Il36rn−/− mice; NET formation was confirmed pathologically. Several cytokines and chemokines were upregulated in the skin lesions of Il36rn−/− mice and Cl-amidine treatment improved these psoriasis-like lesions. These results suggest that NET formation plays an important role in the pathology of psoriasis-like lesions in these mice and might represent a promising therapeutic target for DITRA.

The roles of T cells, macrophages, and DCs have been reported in the pathogenesis of psoriasis vulgaris [15][16][17][18][19] . However, the role of neutrophils in this pathology have gradually been clarified 20 but have not been sufficiently elucidated. Neutrophils play important roles in many diseases, including infectious and neoplastic, autoimmune, and chronic inflammatory diseases [21][22][23][24][25] . Upon activation, neutrophils undergo a cell death process known as NETosis, in which nuclear substances are extruded into the extracellular space 26,27 . These structures are named neutrophil extracellular traps (NETs) and are large, web-like structures composed of granule proteins, histones, and decondensed DNA 28 . The existence of NETs has been reported in psoriatic skin, where they might play a role in inducing increased expression of human β-defensin-2 29 . Therefore, NETs and neutrophils can induce inflammation through various mechanisms, including inflammasome activation 30 , the triggering of Toll-like Scientific Reports | (2020) 10:20149 | https://doi.org/10.1038/s41598-020-76864-y www.nature.com/scientificreports/ receptor 7 (TLR7) and TLR9 via self-antigen complexes such as cathelicidin antimicrobial peptide LL37 DNA 31 , macrophage pyroptosis stimulation 32 , and IL-36 cytokine processing and activation 33,34 .
Here, we aimed to clarify the role of NET signaling in DITRA and develop an effective therapy for IMQinduced severe psoriasis in Il36rn −/− mice. As NETs might be involved in DITRA, it could be possible to prevent the development of severe psoriasis-like lesions by prohibiting their formation. Therefore, this study was conducted to determine the immunological pathology associated with severe psoriasis-like lesions in mice with DITRA.

Results
Estimation of psoriasis area and severity index (PASI) scores and histological characteristics in Il36rn −/− mice after consecutive topical application of IMQ cream for 3 days. IMQ treatment for 3 consecutive days induced psoriasis-like lesions in Il36rn −/− mice. For the purpose of assessing the severity of this psoriasis-like lesion, we evaluated the eruption using the PASI score 35 and compared Il36rn −/− and wild-type mice (n = 6). Il36rn −/− mice showed a significant increase in PASI scores ( Fig. 1A-C). Severe scaling was observed in IMQ-induced Il36rn −/− mice. Next, we pathologically estimated epidermal area, the number of infiltrated neutrophils and NET formation. Il36rn −/− mice showed a significant increase in the epidermal area ( Fig. 2A,B) compared to that in wild-type mice after IMQ treatment for 3 consecutive days. To evaluate inflammatory cell infiltration in IMQ-induced psoriasis lesions, immunostaining for CD11c, F4/80, CD3, and myeloperoxidase (MPO) was performed. Il36rn −/− mice showed a significant increase in CD11c-, CD3-, and MPO-positive cells compared to those in wild-type mice (Fig. 2C). In addition, to evaluate NET formation in IMQ-induced psoriasis-like lesions, immunofluorescent co-staining for MPO and citrullinated histone H3 was Chemokine and cytokine expression in psoriasis-like lesions in Il36rn −/− mice. The expression of genes encoding tumor necrosis factor (TNF)-α, C-C motif chemokine ligand 4 (CCL4), CCL5, C-X-C motif chemokine ligand 1 (CXCL1), CXCL2, IL-1β, IL-6, IL-17A, IL-23p19, and IL-36γ after IMQ treatment for 3 consecutive days was measured via real-time reverse transcription polymerase chain reaction (RT-PCR) in Il36rn −/− and wild-type mice (Fig. 3). Il36rn −/− mice showed significantly increased TNF-α, CXCL1, CXCL2, IL-1β, IL-17A, and IL-36γ expression levels compared to those in wild-type mice. In contrast, the loss of IL-36Ra did not reach significant difference in a significant difference in CCL4, CCL5, IL-6, or IL-23p19 mRNA expression levels in comparison to those in wild-type mice.
In vitro macrophage and NET culture. In our study, Il36rn −/− mice showed significantly increased IL-36γ expression levels compared to those in wild-type mice (Fig. 3). It has been reported that NETs stimulate monocytes and induce secretion of IL-1β, IL-6, IL-23, and TNF-α 40 . In addition, the activation of monocytes leads to cell maturation with an increase in Th17 cells, leading to IL-36γ production by keratinocytes through T-cell mediated immune reactions via the IL-17/IL-23/IL-22 axis 40,41 . Therefore, it is possible that monocytes activated by NETs induce the secretion of IL-36γ through T-cell mediated immune reactions via the IL-17/ IL-23/IL-22 axis. However, it has been reported that macrophages also play an important role in the pathogenesis of psoriasis 19 and that NETs stimulate IL-1β and IL-18 release by macrophages derived from lupus patients 30 .
In addition, it has been reported that NETs promote peritoneal macrophage pyroptosis, a caspase-1-dependent  www.nature.com/scientificreports/ regulated cell death, and the production of macrophage-derived Il-1β and TNF-α 32 in sepsis. Furthermore, it is also known that IL-36 produced by macrophages is involved in the pathological condition in psoriasis, rheumatoid arthritis, and Crohn's disease 42 . Therefore, we hypothesized that the increase of IL-36γ in Il36rn −/− mice is produced by macrophages activated by NETs. To test this possibility, we performed co-culture of macrophages with NETs, and measured mRNA expression levels of TNF-α, IL-1β, and IL-36γ, which are involved in the pathological condition of psoriasis. Peritoneal macrophages of wild-type mice and Il36rn −/− mice were used for in vitro analysis. IL-1β, IL-36γ, and TNF-α mRNA expression levels were significantly increased in NET-added macrophages compared to those in macrophages alone, and these mRNA increases were inhibited by pre-treatment of neutrophils with Cl-amidine (Fig. 5). There was no significant difference in mRNA expression levels between macrophages derived from Il36rn −/− mice and those from wild-type mice. Thus, the production of IL-1β, IL-36γ, and TNF-α by macrophages was increased by NET stimulation in vitro.

Discussion
The results of our study demonstrate that IMQ treatment for 3 consecutive days induced psoriasis-like lesions in Il36rn −/− mice (Fig. 1A,B). The severity of the disease is aggravated and accelerated in Il36rn −/− mice compared to that in wild-type mice. Many neutrophils infiltrated into the superficial dermis and epidermal thickness and the number of infiltrated MPO-positive neutrophils were significantly increased in Il36rn −/− mice compared to those in wild-type mice ( Fig. 2A,B,C), which is consistent with previous studies 13 . In addition, real-time RT-PCR analysis showed that IL-1β, IL-17A, IL-36γ, CXCL1, and CXCL2 expression was increased in IMQ-induced psoriasis-like lesions in Il36rn −/− mice (Fig. 3). As these results were indicative of the global gene expression occurring in the skin, the source of these genetic changes remains unknown. It has been reported that various immune cells and cytokines are involved in the pathogenesis of IMQ-induced psoriasis-like lesions [15][16][17][18][19][20] . TLR7 stimulation by IMQ causes the production of IL-23 and IL-36 from DCs 14 and early IL-23 production is caused by IL-36 signaling in keratinocytes 43 . The αβ T cells or γδ T cells, which are main producers of IL-17 in IMQinduced psoriasis-like lesions, got activated by IL-23, and secrete IL-17 and IL-22, resulting in the proliferation of keratinocytes 14 . IL-36 derived from DCs stimulates themselves and keratinocytes to further produce IL-36. Additionally, activated keratinocytes produce CXCL1 and CCL20 to facilitate the migration of neutrophils, αβ T cells, and γδ T cells 14 . Although the presence of γδ cells was not demonstrated in Il36rn −/− mice, considering previous reports, our results might indicate that stimulation by IL-36 derived from DCs causes the proliferation of keratinocytes and that various chemokines produced by keratinocytes induce the migration of neutrophils, αβ T cells, and γδ T cells. However, the difference in IL-23p19 expression in skin lesions did not reach significance in Il36rn −/− mice compared to that in wild-type mice (Fig. 3). It has been reported that not only IL-23 but also IL-1β induces IL-17 production from γδ T cells 44 . As IL-1β mRNA levels in skin lesions were significantly elevated in Il36rn −/− mice compared to those in wild-type mice (Fig. 3), the increase in IL-17A in Il36rn −/− mice would be caused mainly by CCL20 and IL-1β rather than the IL-23/IL-17/IL-22 axis.
Il36rn −/− mice showed a significant increase in the number of infiltrated MPO-positive neutrophils (Fig. 2C), which is consistent with the results of previous studies 13 . In addition, the area of NETs was also increased in Il36rn −/− mice compared to that in wild-type mice (Fig. 2E). It has been reported that NETs activate keratinocytes and promote the production of LCN2, IL-36γ, CXCL8, and CXCL1 20 . Endogenous neutrophil-derived TLR4 ligands synergize with IL-36, signaling through MyD88 and NF-κB activation, to induce LCN2 and IL-36γ production. In turn, the upregulated LCN2 modulates NET formation and neutrophil migration, enhancing and sustaining the inflammatory response 20 . In accordance with a previous report, IL-36γ and CXCL1 were increased in IMQ-induced psoriasis-like lesions in Il36rn −/− mice, abundant with NETs (Fig. 3). Next, we examined the effect of Cl-amidine, which is a pan-PAD inhibitor, in IMQ-induced psoriasis-like lesions of Il36rn −/− mice. The administration of Cl-amidine significantly inhibited NET formation in Il36rn −/− mice. In addition, PASI scores, the epidermal area, and the number of infiltrated MPO-positive cells were significantly reduced by Cl-amidine www.nature.com/scientificreports/ administration compared with those measured in vehicle-treated Il36rn −/− mice ( Fig. 4B and C). Furthermore, Cl-amidine-treated Il36rn −/− mice with psoriasis-like lesions showed decreased TNF-α, CXCL1, IL-1β, IL-36γ, and IL-17A expression compared to those in untreated, IMQ-induced Il36rn −/− mice. Thus, the inhibition of NET formation improved IMQ-induced psoriasis-like lesions in Il36rn −/− mice. Since Cl-amidine is a pan-PAD inhibitor, it is therefore possible that the effect of Cl-amidine on Il36rn −/− mice is due to the inhibition of PADs other than PAD4. PAD1 mutations have been associated with the severity of psoriasis in patients 45 , and PAD1 is importantly expressed in skin keratinocytes and hair follicles 46 . Although the effect of Cl-amidine could affect PAD1 in skin keratinocytes and hair follicles, Shao et al. also reports that the administration of Cl-amidine to IMQ-induced psoriasis-like lesions is as effective as DNase I, which breaks down NETs, in scaling, acanthosis, and inflammatory infiltration 20 . As this information is merely comparison of the effect between Cl-amidine and DNase I, it strongly suggests that the inhibition of PAD4 by Cl-amidine suppressed NET formation and improved IMQ-induced psoriasis-like lesions in Il36rn −/− mice. Collectively, increased MPO-positive neutrophil infiltration and NET formation in Il36rn −/− mice would play a central role in the pathogenesis of IMQ-induced psoriasis-like lesions. NETs promote macrophage pyroptosis and the production of macrophage-derived Il-1β and TNF-α 32 . In addition, it has been reported that macrophages and monocytes secrete various cytokines such as IL-36 42 . Therefore, we investigated the changes in IL-1β, IL-36γ, and TNF-α production by macrophages upon NET stimulation. IL-1β, IL-36γ, and TNF-α mRNA expression levels were significantly increased in NET-added macrophages compared to those in macrophages alone (Fig. 5). These mRNA increases were inhibited by pre-treatment of Cl-amidine (Fig. 5). These results suggest that various cytokines involved in the pathogenesis of IMQ-induced psoriasis-like lesions would be produced not only by keratinocytes but also by macrophages and that NETs could promote the production of these cytokines.
Based on our results and previous reports, the mechanisms of IMQ-induced psoriasis-like lesions in Il36rn −/− mice are illustrated in Fig. 6. DCs activated by IMQ secrete IL-36γ, causing the proliferation of keratinocytes. CCL20 generated from the keratinocytes mainly enhances the migration of γδ T cells and the secretion of a large amount of IL-17A. Neutrophils can cause NETosis, and cytokines, including IL-1β, IL-36γ, and TNF-α ,are secreted by macrophages activated by NETs; moreover, MyD88/NF-kB activation by IL-36γ and the production of IL-17A from γδ T cells promoted by IL-1β form a pathological condition of IMQ-induced psoriasis-like lesions.
In summary, our results suggest that IL-36Ra loss causes the development of severe psoriasis-like lesions after IMQ treatment for 3 consecutive days by increasing the infiltration of neutrophils into the skin, which is associated with the activation of IL-36R-mediated sustained inflammatory signaling. NET formation also contributes to the development of severe psoriasis-like lesions in Il36rn −/− mice. Therefore, NETs may represent promising therapeutic targets for DITRA.

Methods
Mice. Il36rn −/− mice were generated as previously reported 11 . All experiments were repeated twice using fertile and healthy mice that displayed no evidence of disease or infection. All mice were housed in a pathogen-free barrier establishment and were screened regularly for pathogens. Male C57BL/6 N (Charles River Laboratories, Inc., Wilmington, MA, USA) mice aged 8-12 weeks were used in this study. All studies and procedures were Psoriasis model in mice. The back skin of male wild-type or Il36rn −/− mice was shaved using an electric clipper and depilatory cream before treatment. To establish a mouse model of psoriasis, wild-type and IL36rn −/− mice were topically administered IMQ cream (62.5 mg Beselna Cream, containing 5% IMQ; kindly gifted by MOCHIDA PHARMACEUTICAL CO., LTD., Tokyo, Japan) on the back skin for 3 consecutive days over an area of 3 × 2 cm (Fig. 1A). In some experiments, Cl-amidine (10 mg/kg/day, Cayman Chemical Company, Ann Arbor, MI, USA), a PAD4 inhibitor, was subcutaneously injected.
Histological assessment. Mice were sacrificed via cervical dislocation, and the fresh back skin of each mouse was harvested. Fresh mouse skin samples were fixed in 4% paraformaldehyde solution for 24 h, dehydrated, and embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin. Epidermal thickness was evaluated by measuring the area of the epidermis. The area of the epidermis within a distance of 10 mm was measured using ImageJ software (version 1.53; NIH, Bethesda, MD, USA, https ://image j.nih.gov/ij/) 47 . Neutrophil infiltration was assessed by counting the number of neutrophils in nine high-power fields per section. Each section was examined independently by two investigators in a blinded manner, and the means of these measurements were used for analysis. For immunohistochemistry of mouse samples, 6-μm-thick sections of paraffin-embedded tissues were cut, deparaffinized in xylene, and rehydrated in phosphate buffered saline (PBS). Deparaffinized sections were treated with endogenous peroxidase blocking solution (horse serum diluted 1:1 in buffer: PBS + bovine serum albumin 1%) for 15 min at room temperature. Sections were then incubated overnight at 4 °C with rabbit monoclonal antibodies (mAb) specific for CD3 (1:500, Cell Signaling Technology, Inc., Tokyo, Japan), and F4/80 (1:800, Cell Signaling Technology, Inc.). Sections were then washed in PBS buffer and biotin-conjugated secondary antibodies were applied, followed by incubation with VECTASTAIN Elite ABC Kit (Vector Laboratories, Inc., Burlingame, CA, USA) for 30 min at room temperature and three washes with PBS for 15 min each. Peroxidase activity was observed using an ImmPACT DAB Substrate Kit (Vector Laboratories, Inc.) and samples were counterstained with hematoxylin. For a negative control, primary antibody was not added to the sections.

RNA extraction and real-time RT-PCR.
Qiagen RNeasy spin columns (QIAGEN, Valencia, CA, USA) was used to extract total RNA from the mouse back skin tissue samples 11 . The total RNA was reverse-transcribed into cDNA using a Prime Script RT Reagent Kit (Takara Bio Inc., Otsu, Japan). Expression levels of genes encoding IL-1β, IL-6, IL-17A, IL-36γ, CCL4, CCL5, CXCL1, CXCL2, TNF-α, and IL-23p19 were measured via realtime RT-PCR using a Light Cycler System (F. Hoffman-La Roche, Ltd, Basel, Switzerland) 11 . PCR samples were prepared in microcapillary tubes as 20-µL reactions containing diluted cDNA solution (2.0 µL), and the PCR program was performed according to the manufacturer's instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as a standard against which the relative mRNA expression levels of different target genes were calculated using the 2 −∆∆Ct method. Primer sequences used for each gene were provided with the pre-verified Primetime qPCR Assay (Integrated DNA Technologies, Inc., Coralville, IA, USA) 12 .
In vitro experiments. Collection of peritoneal cells from the peritoneal cavities of mice. Mice were intraperitoneally injected with cold PBS (10 mL). The abdomen was massaged to suspend the cells, and 8 mL PBS was collected. The cells obtained were centrifuged at 500 × g at 4 °C for 10 min. Red blood cell lysis buffer was added, and the cells were again centrifuged at 500 × g at 4 °C after incubation at 4 °C for 10 min to obtain the peritoneal cells 48 .
Collection of neutrophils from the peritoneal cavities of mice. Neutrophils were collected from the peritoneal cavities of mice using a Neutrophil Isolation Kit (Cayman Chemical Company) according to the manufacturer's Scientific Reports | (2020) 10:20149 | https://doi.org/10.1038/s41598-020-76864-y www.nature.com/scientificreports/ protocol. Casein (7.5%, 1 mL) was injected into the peritoneal cavity of mice followed by neutrophil isolation medium (5 mL, PBS + 1% BSA) 24 h later. The abdomen was massaged to suspend the cells, and the medium (4 mL) was collected. The fluid from the peritoneal cavity was slowly layered onto 63% Percoll solution, which was then centrifuged at 1,000 × g at 27 °C for 20 min. The Percoll solution was aspirated, and PBS with BSA (9 mL) was added. The samples were centrifuged at 500 × g at 27 °C for 5 min. Red blood lysis buffer (5 mL) was added, and the samples were centrifuged at 500 × g at 27 °C for 10 min after incubation at 27 °C for 10 min to obtain the mouse neutrophils.
NET generation. For NET generation, 1 × 10 6 neutrophils were seeded into 24-well plates in serum-free Dulbecco's modified Eagle's medium (DMEM) and then stimulated to release NETs by adding phorbol 12-myristate 13-acetate (50 nM; Sigma-Aldrich Co. LLC., St Louis, MO, USA) for 4 h at 37 °C. The medium was removed, and the cell layer was carefully washed with PBS (2 mL). The PBS was collected after vigorous agitation and centrifuged at 500 × g at 4 °C for 10 min. Cell-free NET structures were then collected in the supernatant phases 20 . Some neutrophils were pretreated with Cl-amidine (10 μM; Cayman Chemical Company) before NET generation for 2 h at 37 °C 49 .
Macrophage culture. Peritoneal cells (mixture of macrophages, B cells, and T cells, 0.5 × 10 6 ) were cultured in 24-well plates in serum-free DMEM. After 24 h, non-adherent cells (B cells and T cells) were removed by gently washing three times with warm PBS 48 . In some experiments, isolated macrophages were co-cultured with NETs originated from 0.5 × 10 6 neutrophils. After 12 h of culture, total RNA was extracted and subsequently reversetranscribed.
Statistical analysis. Data were analyzed using a GRAPHPAD PRISM software (version 7, Graph Pad Software, La Jolla, CA, USA, https ://www.graph pad.com/scien tific -softw are/prism /) and presented as means ± standard deviations. For comparisons between groups, Mann-Whitney U tests and one-way analysis of variance were used. Values of p < 0.05 were considered to indicate statistical significance.

Data availability
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