RIPK3 promotes skin inflammation by enhancing IL-36α signaling and necroptosis in keratinocytes

Abstract

Psoriasis is a chronic inflammatory skin disease characterized by complex pathogenesis involving multiple factors. Keratinocytes, as key structural components, play a critical role in immune regulation and contribute to disease progression through interactions with various immune cells. Receptor-interacting protein kinase 3 (RIPK3) is well-known for its role in necroptosis, acting alongside RIPK1 and mixed-lineage kinase domain-like (MLKL). While studies have shown that inhibitors of necroptosis could alleviate psoriasis-like skin inflammation, direct genetic evidence of RIPK3 is lacking. Furthermore, recent studies have highlighted RIPK3's independent biological functions beyond necroptosis, yet its pathological role in inflammatory skin disease remains poorly understood. This study aimed to elucidate the pathological role of RIPK3 in the progression of skin inflammation, particularly in keratinocytes. We demonstrated that RIPK3 expression was significantly upregulated in psoriasis patients and mice with imiquimod (IMQ)-induced skin inflammation. Importantly, keratinocyte-specific knockout of RIPK3 using gene-editing tools significantly alleviated IMQ-induced skin inflammation in mice. Interestingly, the absence of RIPK3 not only inhibited necroptosis and associated inflammatory responses but also significantly reduced interleukin-36α (IL-36α) expression in keratinocytes. IL-36α, known to drive skin inflammation, promote immune cell recruitment, and disrupt the epidermal barrier, is a critical mediator of inflammatory skin disease pathogenesis. Further investigation using MLKL-knockout mice and keratinocytes revealed that RIPK3 regulates the IL-36α/NF-κB signaling axis through an MLKL-independent mechanism. Collectively, our findings uncover a dual pathogenic role for RIPK3 in skin inflammation: promoting inflammation through both canonical necroptosis and a distinct, non-necroptotic pathway that drives IL-36α activation. These insights not only identify RIPK3 as a potential therapeutic target for psoriasis-like skin inflammation but also uncover its previously unappreciated roles in inflammatory diseases beyond necroptosis.

Fig. 1. RIPK3 is upregulated in patients with psoriasis and IMQ-treated mice.

A The mRNA expression levels of RIPK3 and MLKL in the skin from normal, non-lesional skin, and lesional skin of psoriasis patients in the Gene Expression Omnibus (GEO) database (GSE13305 and GSE14905). The mRNA expression levels of RIPK3 and MLKL (B) and representative western blot assay of RIPK3 and MLKL (C) in the skin of IMQ-treated Balb/c mice. Normal (n = 6), IMQ (n = 8). D Immunofluorescence analysis of RIPK3 and p-MLKL in skin tissues of IMQ-treated Balb/c mice. Scale bars, 100 μm. All dates are shown as means ± SEM. ^*P < 0.05, ^***P < 0.001, compared as indicated, were measured by one-way ANOVA or Student’s t-test.

Fig. 2. RIPK3^ΔKC alleviates the severity of IMQ-induced skin damage.

A Representative photographs of the back skin in each group (Day 5). B Daily thickening, erythema, scaling scores (0-4) and total scores (0-12) in each group. Representative H&E staining images (C) and epidermal thickness (D) in each group. Scale bars, 250 μm. Representative splenic images (E) and splenic index (F) in each group. Representative western blot assay (G) and quantification (H) of RIPK3 and MLKL in the dorsal skin. RIPK3^f/f (n = 5), RIPK3^f/f IMQ (n = 7), RIPK3^ΔKC (n = 4), RIPK3^ΔKC IMQ (n = 6). All dates are shown as means ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, compared as indicated, were measured by one-way or two-way ANOVA. There was a significant difference between the RIPK3^f/f and RIPK3^f/f IMQ groups.

Fig. 3. RIPK3^ΔKC mitigates skin inflammation in IMQ-treated mice.

A The level of cytokines (TNF-α, IL-6 and IFN-γ) in serum was measured by ELISA. B Heatmap showing mRNA expression levels of cytokines, chemokines and antimicrobial peptides in the skin as measured by RT-PCR. C Percentage of leukocytes (CD45⁺) and neutrophils (CD11b⁺Gr-1⁺) cells analyzed by flow cytometry in the skin. Immunofluorescence detection of protein levels of S100A8 (D) and Gr-1 (E) in the skin. Scale bars, 100 μm. RIPK3^f/f (n = 5), RIPK3^f/f IMQ (n = 7), RIPK3^ΔKC (n = 4), RIPK3^ΔKC IMQ (n = 6). These results are representative of three independent experiments. All dates are shown as means ± SEM, compared as indicated, were measured by one-way ANOVA.

Fig. 4. Targeting RIPK3 suppresses TSZ-induced keratinocyte necroptosis and inflammation.

Western blot (A) and RT-PCR (B) analysis of the expression level of RIPK3 in primary keratinocytes from RIPK3^f/f and RIPK3^ΔKC mice. C Cell viability of RIPK3-deficient primary keratinocytes following TSZ treatment was detected by CCK-8 kit. D Representative western blot analysis of the necroptosis pathway after TSZ treatment for 12 hours in primary keratinocytes. E Representative immunofluorescence analysis of Occludin in primary keratinocytes after TSZ treatment for 6 hours in primary keratinocytes. Scale bars, 100 μm. F RT-PCR analysis of mRNA levels of chemokines, cytokines and antimicrobial peptides after TSZ treatment for 6 hours in primary keratinocytes. G Cell viability of HaCaT cells treated with GSK’2982772 (100, 10 and 1 nM) and GSK’872 (10, 5 and 2.5 μM) was detected by CellTiter kit. Representative western blot analysis of the necroptosis pathway (H) in HaCaT cells treated with TSZ for 24 hours, and tight junction proteins (I) in HaCaT cells treated with TSZ for 6 hours. Representative immunofluorescence (J) and mRNA expression levels of IL-1α, IL-1β and CXCL8 measured by RT-PCR (K) in HaCaT cells treated with TSZ for 6 hours. Scale bars, 100 μm. These results are representative of three independent experiments. All dates are shown as means ± SEM. ^***P < 0.001, compared as indicated, ^###P < 0.001, compared with BLANK, were measured by Student’s t-test, one-way or two-way ANOVA. There was a significant difference between the RIPK3^f/f and RIPK3^f/f TSZ groups.

Fig. 5. RIPK3 amplifies IL-36 signaling in skin inflammation.

A Correlation between IL-36α and RIPK3 expression in psoriasis patients in the GEO database. GSE13305: normal (n = 64, green), non-lesional skin (n = 58, blue) and lesional skin (n = 58, red); GSE14905: normal (n = 21, green), non-lesional skin (n = 28, blue) and lesional skin (n = 33, red). The mRNA expression of Il-36α (B); representative western blot (C) analysis of IL-36α protein levels and immunofluorescence staining (D) of IL-36α levels in the skin. Scale bars, 100 μm. The mRNA expression of IL-36α in primary keratinocytes from RIPK3^f/f and RIPK3^ΔKC mice (E) and normal mice (F) treated with TSZ for 6 hours. G Representative western blot assay of IL-36α pathway in primary keratinocytes treated with IL-36α for 15 minutes. H The mRNA expression of Ccl20, Cxcl2, Cxcl10 and Il-36γ in primary keratinocytes treated with IL-36α for 6 hours. These results are representative of three independent experiments. All dates are shown as means ± SEM. ^*P < 0.05, ^***P < 0.001, compared as indicated, ^###P < 0.001, compared with BLANK, were measured by one-way or two-way ANOVA. Significant differences were observed between the RIPK3^f/f and RIPK3^f/f IMQ groups, the RIPK3^f/f and RIPK3^f/f TSZ groups, and the RIPK3^f/f and RIPK3^f/f IL-36α groups.

Fig. 6. RIPK3 promotes IL-36α signaling in a MLKL-independent manner in keratinocytes.

A Representative western blot assay of necroptotic proteins in primary keratinocytes from RIPK3^f/f and RIPK3^ΔKC mice treated with IL-36α for 6 hours. B RT-PCR detection of mRNA expression levels of S100a8, S100a9, Il-1α and Il-36α after TSZ induction in primary keratinocytes. C Representative western blot images assay of necroptotic proteins in IL-36α-induced primary keratinocytes. D RT-PCR detection of mRNA expression levels of cytokines such as Cxcl2, Lcn2, S100a9 and Il-36α after IL-36α induction in primary keratinocytes. E Representative images of mice treated with saline and IL-36α at day 5 to demonstrate the scaling of the treated area. Ear thickness (F) and ear swelling (G) in mice treated with saline and IL-36α in each group. n = 5 mice per group. These results are representative of three independent experiments. All dates are shown as means ± SEM. ^***P < 0.001, ns indicates no significance, compared as indicated, were measured by two-way ANOVA. Significant differences were observed between the WT and WT TSZ groups, as well as between the WT and WT IL-36α groups.

Fig. 7. RIPK3 promotes skin inflammation by enhancing IL-36α signaling and necroptosis in keratinocytes.

In keratinocytes, RIPK3 orchestrates skin inflammation through a dual pathogenic mechanism. It initiates MLKL-dependent necroptosis, releasing DAMPs like IL-36α. This extracellular IL-36α, in turn, activates the pro-inflammatory NF-κB signaling axis. Beyond merely initiating this cascade, RIPK3 critically functions in an MLKL-independent manner to directly potentiate NF-κB activation, driving robust expression of inflammatory mediators (e.g., CCL20, CXCLs). This interplay constitutes a powerful feed-forward loop that amplifies and sustains cutaneous inflammation.