Blockade of IL-18Rα-mediated signaling pathway exacerbates neutrophil infiltration in imiquimod-induced psoriasis murine model

Abstract

Psoriasis is an immune-mediated inflammatory disease of the skin, which is characterized by epidermal hyperkeratosis and neutrophil infiltration. The interleukin (IL)-17/IL-23 pathway and associated cytokines play major roles in the pathogenesis and exacerbation of psoriasis. The IL-18/IL-18 receptor (R) α signaling pathway is important for Th1 cytokine production and differentiation of Th1 cells; however, its role in the pathogenesis of psoriasis remains unknown. In this study, we investigated the effect of the IL-18Rα-mediated signaling pathway in the pathogenesis of psoriasis in Il18ra-deficient mice (Il18ra^-/-) and wild-type imiquimod (IMQ)-induced psoriatic dermatitis model mice. Blocking this pathway exacerbated IMQ-induced psoriatic skin inflammation. Il18ra deficiency led to significant increases in the levels of IL-1β, IL-6, IL-8, IL-17A, IL-23, and chemokine (C-X-C motif) ligand 2 in skin lesions. Gr1-positive cells highly infiltrated psoriatic skin lesions in Il18ra^-/- mice compared to those in wild-type mice. Citrullinated histone H3-positive area was relatively broad in Il18ra^-/- mice. These results suggest that IL-18Rα-mediated signaling pathways may inhibit psoriatic skin inflammation by regulating infiltration and activation of neutrophil and other innate immune cells.

Keywords: IL-18Rα; immune-mediated inflammatory disease; innate immunity; neutrophil; psoriasis.

Figure 1

Experiment schedule in wild-type and Il18ra^−/− mice with psoriasis induced by IMQ. The IMQ cream (5%) or petroleum was applied to the shaved back skin of female Il18Ra^−/− and WT C57BL/6 mice aged 8–11 weeks consecutively for 5 days (day 0–4). The number of Il18ra^−/− applied IMQ and petroleum, and WT applied IMQ and petroleum were 15, 8, 12 and 9. Il18ra, interleukin-18 receptor alpha; WT, wild type; IMQ, imiquimod.

Figure 2

IMQ-induced psoriasis was exacerbated in Il18ra deficiency mice were treated with imiquimod cream or petroleum for 5 days (A). Cumulative scores, as PASI score for mice, and each independent score (erythema, thickening, scaling) of Il18ra^−/− and WT were scored consecutively for 5 days (B). Cutaneous involvement on the back of IL-18Rα^−/− or WT mice treated with IMQ or petroleum. Photographs were taken on day 5. Values are mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Il18ra, interleukin-18 receptor alpha; WT, wild type; IMQ, imiquimod; ns, not significant.

Figure 3

Il18ra-mediated signaling pathway suppresses antigen presenting cells (APCs) activities in IMQ-induced psoriasis hematoxylin and eosin (H&E) stained specimens of Il18ra^−/− (n = 15 and 8) and WT (n = 12 and 9) skin sections on day 5 after the start of treatment with IMQ or petroleum (A–D). Arrows indicate skin thickness. Skin of IL-18Rα^−/− was thicker than WT (E). Increased F4/80 and CD11c positive cell infiltration in Il18ra^−/− (F,I: n = 7) than WT (G,J: n = 7) skin tissues. The Il18ra^−/− group had significantly higher F4/80 positive (H) and CD11c positive (K) cells. Conversely, the WT group had significantly higher CD4 positive cells than the Il18ra^−/− group (L–N: n = 7). Photomicrographs were taken at 10× magnification. Values are expressed as mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Il18ra, interleukin-18 receptor alpha; WT, wild type; IMQ, imiquimod.

Figure 4

Il18ra-mediated signaling pathway suppresses the systemic inflammatory response independent of the Th1 response. Systemic inflammation indicators such as serum IL-17A and IL-6 are highly upregurated in the Il18ra^−/− group than in the WT group (A,B). Whereas, the adaptive immune response characterized by IFN-γ production is more downregulated than in the WT group (C,D). The data are expressed as mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. IL-6, interleukin-6; IL-17A, interleukin-17A; Il18ra, interleukin-18 receptor alpha; WT, wild type; IFN-γ, interferon gamma; WT, wild type; IMQ, imiquimod.

Figure 5

Effects of Il18ra-mediated signaling on cytokines and chemokines in dermatitis. Gene expression levels of cytokines, chemokines, transcription factors, Toll-like receptor and inflammasome related proteins in skin lesions were measured by real time PCR. The expression levels were normalized to the expression of Rn18s and are expressed relative to the values in untreated control mice. The expression in skin lesions of cytokines involved in the pathogenesis of psoriasis, such as I17a and Il23a, and chemokines that induce neutrophils to enter lesions, such as Cxcl2, was significantly increased in the IL18ra^−/− group. In addition, there were also differences in the enhancement of cytokines such as Il1β and Nlrp3, which are involved in the process and outcome of inflammasome activation, suggesting that their activation may be more strongly involved in the exacerbation of dermatitis in the IL18ra^−/− group than in the WT. The data are expressed as mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001. Il1β, interleukin-1 be-ta; Il4, interleukin-4; Il6, interleukin-6; Il17a, interleukin-17a; Il18, interleukin-18; Il18ra, interleukin-18 receptor alpha; Il22, interleukin-22; Il23a, interleukin-23a; Cxcl2, chemokine (C-X-C motif) ligand 2; Nlrp3, nucleotide-binding domain and leucine-rich repeat pyrin containing protein-3; Ifng, interferon gamma; Tlr4, Toll like receptor 4; Rorc, retinoic acid receptor-related orphan receptor-gamma t; Foxp3, Forkhead box protein 3; WT, wild type; IMQ, imiquimod.

Figure 6

Il18ra-mediated signals may suppress IL-17 production by neutrophils activation. Infiltration of Gr1 positive cells and citrullinated histon 3 (NETs) into skin tissues of Il18ra^−/− applied IMQ (A,B). Photomicrographs were taken at 10× magnification to compare the area of Gr1 positive cells and NETs infiltration into skin lesions between Il18ra^−/− and WT mice treated with IMQ. Increased infiltration of Gr1 positive cells and NETs in Il18ra^−/− were observed (C,D: n = 7). Gr1 positive cell infiltration and serum IL-17A levels correlate moderate and NETs and Il17a mRNA levels in skin lesion also have correlation (Gr1, R² = 0.4480, p = 0.0173; NETs, R² = 0.6828, p = 0.0009) (E,F: n = 12). The data are expressed as mean ± SD. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001. IL-17A, interuikin-17A; l18ra, interleukin-18 receptor alpha; NETs, neutrophil extracellular traps; WT, wild type; IMQ, imiquimod.