Neutrophil extracellular traps are involved in enhanced contact hypersensitivity response in IL-36 receptor antagonist-deficient mice

Abstract

Loss-of-function homozygous or compound heterozygous mutations in IL36RN, which encodes interleukin-36 receptor antagonist (IL-36Ra), have been implicated in the pathogenesis of skin disorders. We previously reported that Il36rn^-/- mice exhibit an enhanced contact hypersensitivity (CHS) response through increased neutrophil recruitment. In addition, Il36rn^-/- mice show severe imiquimod-induced psoriatic skin lesions and enhanced neutrophil extracellular trap (NET) formation. We hypothesized that NETs may play an important role in the CHS response. To confirm this, we examined the CHS response and NET formation in Il36rn^-/- mice. Il36rn^-/- mice showed enhanced CHS responses, increased infiltration of inflammatory cells, including neutrophils, CD4⁺ T cells, and CD8⁺ T cells, NET formation, and enhanced mRNA expression of cytokines and chemokines, including IL-1β, C-X-C motif chemokine ligand (CXCL)1, CXCL2, and IL-36γ. Furthermore, NET formation blockade improved the CHS response, which consequently decreased inflammatory cell infiltration and NET formation. Consistently, we observed decreased expression of these cytokines and chemokines. These findings indicate that IL-36Ra deficiency aggravates the CHS response caused by excessive inflammatory cell recruitment, NET formation, and cytokine and chemokine production, and that NET formation blockade alleviates the CHS response. Thus, NET formation may play a prominent role in the CHS response.

Figure 1

Effect of Cl-amidine on the contact hypersensitivity response in wild-type mice (WT) and Il36rn^−/− mice (KO). (a) Representative images of the ear tissues of KO and WT treated with Cl-amidine (10 mg/kg/day) or vehicle 48 h after the DNFB challenge. (b) Measurement of ear thickness in KO and WT treated with Cl-amidine (10 mg/kg/day) or vehicle 24 h and 48 h after the DNFB challenge. All values represent the mean ± SEM obtained from six mice per group. (*p < 0.05, ** p < 0.01).

Figure 2

Effect of Cl-amidine on inflammatory cell infiltration in the ear skin during the contact hypersensitivity response in wild-type mice (WT) and Il36rn^−/− mice (KO). (a) Representative histological images of ear skin from KO and WT treated with Cl-amidine (10 mg/kg/day) or the same quantity of vehicle 48 h after the DNFB challenge (hematoxylin and eosin (H&E) staining, myeloperoxidase (MPO) staining, F4/80 staining, CD4 staining, and CD8 staining; all scale bars = 100 μm). The infiltrated neutrophils tested positive for MPO (red). The infiltrated macrophages were stained for F4/80. CD8+-positive T cells were indicated by arrow heads. (b) The number of MPO⁺ neutrophils (MPO), F4/80⁺ macrophages (F4/80), CD4⁺ T cells (CD4), and CD8⁺ T cells (CD8) per field (0.16 mm²) was counted under a microscope. All values represent the mean ± SEM obtained from six mice per group. (**p < 0.01).

Figure 3

The effect of Cl-amidine on neutrophil extracellular trap (NET) formation during the contact hypersensitivity response in wild-type mice (WT) and Il36rn^−/− mice (KO). (a) Representative immunofluorescence image of NET structures. Arrow heads show the formation NETs that were myeloperoxidase-positive (red) and citrullinated histone-positive (green). The nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; blue). Scale bar = 50 μm. (b) The area of NETs was measured using the ImageJ software. Each histogram shows the relative area of NETs at 48 h after the DNFB challenge. All values represent the mean ± SEM obtained from six mice per group (**p < 0.01).

Figure 4

Effect of Cl-amidine on the mRNA expression levels in the skin during the contact hypersensitivity response in wild-type mice (WT) and Il36rn^−/− mice (KO). Relative mRNA expression of interleukin (IL)-1β, IFN-γ, IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor alpha (TNF-α), C-X-C motif chemokine ligand (CXCL) 1, CXCL2, IL-36α, IL-36β, and IL-36γ was quantified using real-time reverse transcription polymerase chain reaction and normalized to endogenous glyceraldehyde-3-phosphate dehydrogenase mRNA expression levels. All values represent the mean ± SEM obtained from six mice per group (*p < 0.05, **p < 0.01).

Figure 5

Scheme illustrating the pathology of contact hypersensitivity lesions in the elicitation phase. In Il36rn^−/− mice, the enhancement of NET formation promoted the production of cytokines, such as interleukin (IL)-36γ and IL-1β, by macrophages. IL-1β may induce IL-17A production from γδ T cells and contribute to neutrophil generation. In addition, IL-36γ stimulation may induce keratinocyte activation, and C-X-C motif chemokine ligand (CXCL) 1 produced by keratinocytes may induce the migration of neutrophils, CD4⁺ T cells, CD8⁺ T cells, and γδ T cells, further promoting neutrophil generation. KC, keratinocyte; DC, dendritic cell; NETs, neutrophil extracellular traps; Neu, neutrophils. γδ T: γδ T cells.