IL-17A-driven psoriasis is critically dependent on IL-36 signaling

Abstract

Plaque psoriasis is an autoinflammatory and autoimmune skin disease, affecting 1-3% of the population worldwide. Previously, high levels of IL-36 family cytokines were found in psoriatic skin lesions, thereby contributing to keratinocyte hyperproliferation and infiltration of immune cells such as neutrophils. While treatment with anti-IL36 receptor (IL36R) antibodies was recently approved for generalized pustular psoriasis (GPP), it remains unclear, if targeting the IL36R might also inhibit plaque psoriasis. Here we show that antibody-mediated inhibition of IL36R is sufficient to suppress imiquimod-induced psoriasis-like skin inflammation and represses the disease's development in a model that depends on IL-17A overexpression in the skin. Importantly, treatment with anti-IL36R antibodies inhibited skin inflammation and attenuated psoriasis-associated, systemic inflammation. This is possibly due to a widespread effect of IL36R inhibition, which not only suppresses pro-inflammatory gene expression in keratinocytes, but also the activation of other immune cells such as T-cells or dendritic cells. In conclusion, we propose that inhibition of the IL-36 signaling pathway might constitute an attractive, alternative approach for treating IL-17A-driven psoriasis and psoriasis-linked comorbidities.

Keywords: IL-17A; anti-IL36R; imiquimod; keratinocytes; psoriasis; spesolimab; systemic inflammation.

Figure 1

IL-36 signaling is upregulated in psoriatic skin lesions of mice and psoriasis patients. (A) mRNA expression levels of the IL36 receptor (IL36R/IL1RL2) in skin samples of 64 healthy individuals and in non-lesional and lesional samples from 58 psoriasis vulgaris patients (GDS4602). Significance was calculated using a 2-tailed Student’s t-test: ** p < 0.01**** < 0.0001. (B) mRNA expression levels of psoriasis-associated cytokines in lesional and non-lesional skin of 85 patients (GDS4600). Significance was calculated using a one-way ANOVA test. ns = not significant, **** p < 0.0001. (C, D) Gene expression of IL-36 cytokines and Il1rl2 in 7 days IMQ-treated skin (C) or lesions of K14-IL17A^ind mice (D). Shown are the relative mRNA levels normalized to β-Actin. Significance was calculated using a 2-tailed Student’s t-test: * p < 0.05, ** p < 0.01, *** < 0.001, ns, not significant. Shown is the mean ± SEM of n = 3 (C) or n = 9-11 (D) animals.

Figure 2

Anti-IL36R treatment suppresses keratinocyte hyperproliferation and infiltration of immune cells in IMQ-induced psoriasis-like skin inflammation. (A) Treatment scheme. Antibody treatment was started 2 days after IMQ treatment. (B) Ear thickness during the course of the experiment (n = 14, except for Ctrl + anti-IL36R n = 6). (C) H&E staining of ears from the treated mice at the endpoint at day 7. Scale: 100 µm. (D) Flow cytometry analysis of skin-infiltrating immune cells from treated animals at day 7 (n = 3 - 5 animals per group). Shown is the relative percentage of positive cells, after pre-gating on viable cells. Significance was calculated using a 2-tailed Student’s t-test: * p < 0.05, ** p < 0.01, *** < 0.001. Shown is the mean ± SEM.

Figure 3

Anti-IL36R treatment suppresses pro-inflammatory cytokine and chemokine expression in IMQ-induced psoriasis-like skin inflammation. (A, B) Gene expression analysis of the skin from Ctrl or IMQ-treated animals that received IgG or anti-IL36R treatment. Shown is the relative gene expression of keratinocyte-derived cytokines and chemokines (A), as well as of psoriasis-associated cytokines deriving from T-cells and myeloid cells (B), normalized to β-Actin. n = 3 - 6. Shown is the mean ± SEM. (C) Protein levels of cytokines and chemokines from ears of IMQ + IgG and IMQ + anti-IL36R-treated animals. Shown are the immunoblot analysis and its evaluation normalized to the reference controls and depicted as relative pixel values. Samples were derived from 3 animals per group and pooled before analysis. Significance was calculated using a 2-tailed Student’s t-test: *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4

Anti-IL36R treatment suppresses skin lesion development and immune cell infiltration in IL-17A-induced psoriasis in vivo. (A) Treatment scheme. (A, C) Body Weight (B) and PASI Score (C) of the animals at the endpoint (15–20 weeks old). (D) Pictures of the animals at endpoint. (E) H&E staining of the ears and skin lesions at the endpoint. Scale: 100 µm (ears) and 200 µm (back skin). (F) Flow cytometry analysis of skin-infiltrating immune cells from treated animals at endpoint (n = 4 - 6 animals per group). Shown is the relative percentage of positive cells, after pre-gating on viable cells. (G) Gene expression analysis of the skin of IgG- or anti-IL36R-treated K14-IL17A^ind mice. Relative gene expression was normalized to Rpl37A. Shown is the gene expression from one treatment group. (H) Protein levels from lesional skin of IgG- and anti-IL36R-treated K14-IL17A^ind mice. n = 3 animals per group. Significance was calculated using a 2-tailed Student’s t-test: * p < 0.05, ** p < 0.01, *** < 0.001, ns, not significant. Shown is the mean ± SEM.

Figure 5

Anti-IL36R treatment inhibits systemic inflammation in K14-IL17A^ind mice. (A) Flow cytometry analysis of the bone marrow from Ctrl mice or K14-IL17A^ind mice that either received IgG or anti-IL36R treatment (n = 2-3 animals per group). The relative percentage of positive cells is shown after pre-gating on viable cells. (B) Oxidative burst was measured in venous blood of control or K14-IL17A^ind mice by analyzing the formation of reactive oxygen and nitrogen species. Blood cells were either restimulated with phorbol-12,13-dibutyrate (PDBu) or with Zymosan A. After addition of the 8-amino-5-chloro-7-phenylpyridol-(3,4-d)pyridazine-1,4-(2H,3H)-dione sodium salt (L-012), the oxidative burst was detected by the created chemiluminescence using a SparkTM Multimode Microplate Reader. n = 4-5. C + D. Gene expression analysis of the spleen (C) and colon (D) from treated mice. Shown is the relative gene expression normalized to β-Actin (C) or Rpl37A (D) for n = 3-4 animals per group. Significance was calculated using a 2-tailed Student’s t-test: * p < 0.05, ** p < 0.01, *** p< 0.001. Shown is the mean ± SEM. (E) H&E staining of the colon at the endpoint. Scale: 100 µm.

Figure 6

Acute suppression of psoriasis-associated inflammation in K14-IL17A^ind mice upon anti-IL36R therapy. (A) Treatment scheme. (B) Pictures of an IgG or anti-IL36R-treated animal at the starting point of the treatment, during the treatment and at the endpoint. (C) PASI Score of the mice before treatment and at the endpoint. (D) H&E staining of the affected skin areas at the endpoint. Scale: 100 µm. (E) Flow cytometry analysis of skin-infiltrating immune cells from treated animals at endpoint (n = 3 - 5 animals per group). Shown is the relative percentage of positive cells, after pre-gating on viable cells. (F) Gene expression of the skin from IgG or anti-IL36R-treated K14-IL17A^ind mice at the endpoint. Relative gene expression was normalized to Rpl37A. Significance was calculated using a 2-tailed Student’s t-test: * p < 0.05, ** p < 0.01, ns, not significant. Shown is the mean ± SEM.