LL37 complexed to double-stranded RNA induces RIG-I-like receptor signalling and Gasdermin E activation facilitating IL-36γ release from keratinocytes

Abstract

The Interleukin-36 (IL-36) cytokine family have emerged as important players in mounting an inflammatory response at epithelial barriers and tailoring appropriate adaptive immune responses. As members of the Interleukin-1 superfamily, IL-36 cytokines lack a signal peptide for conventional secretion and require extracellular proteolysis to generate bioactive cytokines. Although the IL-36 family plays an important role in the pathogenesis of plaque and pustular psoriasis, little is known about the release mechanisms of these cytokines from keratinocytes and the physiological stimuli involved. Nucleic acid released from damaged or dying keratinocytes initiates early inflammatory signals that result in the breaking of tolerance associated with psoriasis pathogenesis onset. Cathelicidin peptide, LL37 binds to DNA or double-stranded RNA (dsRNA) and activates a type I Interferon responses in plasmacytoid dendritic cells and keratinocytes. Here, we demonstrate that LL37 binds to dsRNA and induces IL-36γ release from human primary keratinocytes. LL37/dsRNA complexes activate RIG-I-like Receptor signalling, resulting in Caspase-3 and Gasdermin E (GSDME) cleavage. Subsequent GSDME pore formation facilitates IL-36γ release. This response is magnified by priming with psoriasis-associated cytokines, IL-17A and IFNγ. IL-36γ release in this manner is largely independent of cell death in primary keratinocytes and lacked extracellular proteolysis of IL-36γ. Conversely, transfection of keratinocytes directly with dsRNA synthetic analogue, Poly(I:C) induces NLRP1 inflammasome activation, which facilitates IL-36γ expression and release in a GSDMD-dependent manner. Inflammasome-associated cell death also enables extracellular processing of IL-36γ by the release of keratinocyte-derived proteases. These data highlight the distinct responses triggered by dsRNA sensors in keratinocytes. Depending on the inflammatory context and magnitude of the exogenous threat, keratinocytes will release IL-36γ coupled with cell death and extracellular cleavage or release the inactive pro-form, which requires subsequent processing by neutrophil proteases to unleash full biological activity, as occurring in psoriatic skin. Cytoplasmic sensing of dsRNA in keratinocytes mediates IL-36γ release via caspase activity and GSDM pore formation Keratinocytes release IL-36γ upon stimulation with intracellular dsRNA alone or complexed to the psoriasis-associated cathelicidin anti-microbial peptide LL37. Left: Transfected dsRNA triggers NLRP1 inflammasome assembly and IL-1β release, which can enhance IL-36γ expression, resulting in IL-36γ release and extracellular cleavage by released proteases. Right: LL37/dsRNA complexes activate a MDA5-MAVS pathway facilitating the release of IL-36γ through Caspase-3 activation and GSDME pore formation.

Cytoplasmic sensing of dsRNA in keratinocytes mediates IL-36γ release via caspase activity and GSDM pore formation Keratinocytes release IL-36γ upon stimulation with intracellular dsRNA alone or complexed to the psoriasis-associated cathelicidin anti-microbial peptide LL37. Left: Transfected dsRNA triggers NLRP1 inflammasome assembly and IL-1β release, which can enhance IL-36γ expression, resulting in IL-36γ release and extracellular cleavage by released proteases. Right: LL37/dsRNA complexes activate a MDA5-MAVS pathway facilitating the release of IL-36γ through Caspase-3 activation and GSDME pore formation.

Fig. 1. Intracellular dsRNA analogue Poly(I:C) is a potent inducer of IL-36γ release from human primary keratinocytes.

A–C Human primary keratinocytes (HPKs) were stimulated or transfected with Poly(I:C) or Poly(dA:dT) (1 μg/ml) for 24 h. A Cell lysates and supernatants were subjected to SDS-PAGE and immunoblotting with indicated antibodies. Supernatants were collected and assayed for B IL-36γ release by ELISA or subjected to C lactate dehydrogenase (LDH) assay analysis. D, E HPKs were transfected with Poly(I:C) (1 μg/ml), stimulated with Val-boroPro (VbP, 1 μM), one UVB pulse (0.0875 J/cm²) or DMSO and incubated for 24 h. D Cell lysates and supernatants were subjected to SDS-PAGE and immunoblotting with indicated antibodies. E Supernatants were collected and assayed for IL-36γ release by ELISA. F, G ASC- or GSDMD-deficient N/TERT-1 cell-lines (sgASC / sgGSDMD) were stimulated with LL37/Poly(I:C) (5:5 μg/ml) complexes, VbP (0.5 μM), transfected Poly(I:C) (PIC, 1 μg/ml), or one UVB pulse (0.0875 J/cm²) for 18 h and cell lysates and supernatants were subjected to SDS-PAGE and immunoblotting with indicated antibodies. H HEK293^IL-36γ cells were transfected with GSDMD or empty vector (EV) (1 μg) for 24 h. IL-36γ levels in supernatants were measured by ELISA. Data are presented as (A, D, F, G) representative of three independent experiments, (B, C, E, H) presented as the mean ±S.E.M. of 3–4 independent experiments and analysed with (B, C, E) one-way ANOVA followed by Dunnett’s multiple comparisons test or (H) a two-tailed t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns non-significant, Lipo Lipofectamine 2000, M protein marker, PIC Poly(I:C), sg single-guide RNA.

Fig. 2. LL37/Poly(I:C) complexes trigger inflammasome activation and IL-36γ release.

A–C ASC-deficient N/TERT-1 cells (sgASC) were stimulated with LL37/Poly(I:C) (5:5 μg/ml) complexes or separately (each 5 μg/ml), VbP (0.5 μM), transfected Poly(I:C) (1 μg/ml), or one UVB pulse (0.0875 J/cm²) for 18 h and A cell lysates and supernatants were subjected to SDS-PAGE and immunoblotting with indicated antibodies or supernatants were analysed by ELISA for B IL-36γ and C IL-1β levels. D, E GSDMD-deficient N/TERT-1 cells (sgGSDMD) were stimulated with LL37/Poly(I:C) (5:5 μg/ml) complexes or VbP (1 μM) for 18 h and D cell lysates and supernatants were subjected to SDS-PAGE and immunoblotting with indicated antibodies or E supernatants were analysed by ELISA for IL-36γ levels. F, G HPKs were stimulated with LL37/Poly(I:C) (5:5 μg/ml or 10:10 μg/ml) complexes for 24 h. Supernatants were analysed for the release of F IL-1β and G IL-36γ by ELISA. Data are presented as a representative (A, D) of three independent experiments or are presented (B, C, E–G) as the mean ±S.E.M. of at least 3–4 independent experiments and subjected to (B, C, E) two-way ANOVA followed by Šidák’s multiple comparisons test or (F, G) one-way ANOVA followed by Dunnett’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns non-significant, Lipo Lipofectamine 2000, M protein marker, PIC Poly(I:C), sg single-guide RNA.

Fig. 3. LL37/Poly(I:C) complexes induce IL-36γ release from keratinocytes independent of inflammasome and GSDMD activity.

A HPKs were primed with IL-17A (100 ng/ml) or left unprimed (medium control) for 6 h prior to stimulation with Poly(I:C), Poly(dA:dT), LL37/Poly(I:C) or LL37/Poly(dA:dT) complexes (all 5 μg/ml) for 18 h. Supernatants were analysed for IL-36γ release by ELISA. B, C GSDMD-deficient N/TERT-1 cells (sgGSDMD) were stimulated with IL-17A (100 ng/ml) for 6 h prior to stimulation with LL37/Poly(I:C) complexes (5 μg/ml), VbP (1 μM), or transfected with Poly(I:C) (1 μg/ml) for 18 h. Supernatants were analysed for D IL-36γ or E IL-1β levels by ELISA. D, E HPKs were stimulated with IL-17A (10 ng/ml) and TNF-α (5 ng/ml) for 5 h, followed by inhibitor treatment with SB 203580 (p38i, 20 μM), Belnacasan (Casp1i, 10 μM), or DMSO for 1 h before stimulation with LL37/Poly(I:C) complexes (5 μg/ml), VbP (1 μM), or anisomycin (ANS, 1 μM) for 18 h. Supernatants were measured for D IL-36γ or E IL-1β by ELISA. F HPKs were primed with IL-17A for 5 h, followed by inhibitor treatment with MCC950 or DMSO for 1 h before stimulation with LL37/Poly(I:C) complexes for 18 h. Cells were treated with MCC950 1 h before stimulation. Supernatants were measured for IL-36γ release by ELISA. Data are presented (A–F) as the mean ±S.E.M. of 3 independent experiments and analysed with (A–C, F) two-way ANOVA and Šidák’s or (D, E) two-way ANOVA followed by Dunnett’s multiple comparisons test. *p < 0.05, **p < 0.01, ****p < 0.0001. ns non-significant, Lipo Lipofectamine 2000, M protein marker, sg single-guide RNA.

Fig. 4. LL37/Poly(I:C) complexes activate Caspase-3 and GSDME cleavage in human primary keratinocytes.

A, B HPKs were stimulated with LL37/Poly(I:C) complexes (5 μg/ml) or were transfected with Poly(I:C) (1 μg/ml) for indicated times control treatment (−) indicates Medium or Lipo, respectively. Supernatants and cell lysates were subjected to SDS-PAGE and immunoblotting with indicated antibodies. * indicates non-specific band (C, D) GSDME-deficient N/TERT-1 cells (sgGSDME) were (D) primed with IL-17A (100 ng/ml) or (C) left unprimed for 6 h prior to transfection with Lipo/Poly(I:C) (1 μg/ml), stimulation with LL37/Poly(I:C) complexes (5 μg/ml) or one UVB pulse (0.0875 J/cm²) for 18 h. Data are presented as a representative (A, B) of three independent experiments or are presented (C, D) as the mean ±S.E.M. of 3 independent experiments and analysed with two-way ANOVA and Šidák’s multiple comparisons test. **p < 0.01, ***p < 0.001, ****p < 0.0001. ns non-significant, Lipo Lipofectamine 2000, M protein marker, sg single-guide RNA.

Fig. 5. Priming of keratinocytes induces RLR expression and rapid release of IL-36γ in response to LL37/Poly(I:C) complexes.

A, B HPKs were stimulated with LL37/Poly(I:C) complexes (5 μg/ml) or were transfected with Poly(I:C) (1 μg/ml) for indicated times (control treatment (−) indicates Medium or Lipo, respectively). A Cell lysates or B Triton-soluble fractions and -insoluble fractions were subjected to SDS-PAGE and immunoblotting with indicated antibodies. C, D HPKs were primed with IL-17A (100 ng/ml) and/or IFNγ (10 ng/ml) for 16 h, followed by (D) stimulation with LL37/Poly(I:C) complexes for 8 h. C Cell lysates assay were subjected to SDS-PAGE and immunoblotting with indicated antibodies. D Supernatants were measured for IL-36γ release by ELISA. E HPKs were primed with IL-17A (100 ng/ml) for 6 h prior to liposomal transfection of Poly(I:C) (1 μg/ml) or hpRNA (1 μg/ml) for 18 h. Supernatants were analysed for IL-36γ release by ELISA. F HPKs were primed with IL-17A (100 ng/ml) and IFNγ (10 ng/ml) for 16 h, followed by stimulation with LL37/Poly(I:C) or LL37/hpRNA complexes (both 5 μg/ml) for 8 h. Supernatants were subjected to ELISA to measure IL-36γ levels. Data are presented as a representative (A–C) of three independent experiments or are presented (D–F) as the mean ±S.E.M. of at least 3 independent experiments and are subjected to two-way ANOVA followed by (D) Tukey’s or (E, F) Šidák’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns non-significant, Lipo Lipofectamine 2000, M protein marker.

Fig. 6. RLR/MAVS pathway activation induces Caspase-3 cleavage and GSDME-mediated IL-36γ release.

A MAVS-, B RIG-I- or C MDA5-deficient N/TERT-1 cell-lines (sgMAVS/sgRIG-I/sgMDA5) were primed with IL-17A (100 ng/ml) or medium (unprimed) for 6 h prior to treatment with LL37/Poly(I:C) complexes (5 μg/ml) or transfection with either Poly(I:C) or hpRNA (both 1 μg/ml) for 18 h. Supernatants were analysed for IL-36γ release by ELISA. D MAVS-deficient N/TERT-1 cells (sgMAVS) were stimulated with LL37/Poly(I:C) complexes (5 μg/ml) at indicated times. Control treatment (-) indicates Medium only. Cell lysates were subjected to SDS-PAGE and immunoblotting with indicated antibodies. HEK293 cells were transfected with plasmids encoding empty vector (−), E Caspase-3 or F GSDME with and without MAVS for 24 h. Cell lysates were subjected to SDS-PAGE and immunoblotting with indicated antibodies. G HEK293^IL-36γ cells were transfected with empty vector (−) or GSDME with and without MAVS for 24 h and supernatants were measured for IL-36γ release by ELISA. Data are presented as a representative (D–F) of 3 independent experiments or are presented as mean ±S.E.M. of at least 3 independent experiments and subjected to a A–C two-way ANOVA followed by Šidák’s or G one-way ANOVA followed by Tukey’s multiple comparisons test. **p < 0.01, ***p < 0.001, ****p < 0.0001. ns non-significant. Lipo Lipofectamine 2000, sg single-guide RNA.