ZBP1 causes inflammation by inducing RIPK3-mediated necroptosis and RIPK1 kinase activity-independent apoptosis

Abstract

Z-DNA binding protein 1 (ZBP1) has important functions in anti-viral immunity and in the regulation of inflammatory responses. ZBP1 induces necroptosis by directly engaging and activating RIPK3, however, the mechanisms by which ZBP1 induces inflammation and in particular the role of RIPK1 and the contribution of cell death-independent signaling remain elusive. Here we show that ZBP1 causes skin inflammation by inducing RIPK3-mediated necroptosis and RIPK1-caspase-8-mediated apoptosis in keratinocytes. ZBP1 induced TNFR1-independent skin inflammation in mice with epidermis-specific ablation of FADD by triggering keratinocyte necroptosis. Moreover, transgenic expression of C-terminally truncated constitutively active ZBP1 (ZBP1ca) in mouse epidermis caused skin inflammation that was only partially inhibited by abrogation of RIPK3-MLKL-dependent necroptosis and fully prevented by combined deficiency in MLKL and caspase-8. Importantly, ZBP1ca induced caspase-8-mediated skin inflammation by RHIM-dependent but kinase activity-independent RIPK1 signaling. Furthermore, ZBP1ca-induced inflammatory cytokine production in the skin was completely prevented by combined inhibition of apoptosis and necroptosis arguing against a cell death-independent pro-inflammatory function of ZBP1. Collectively, these results showed that ZBP1 induces inflammation by activating necroptosis and RIPK1 kinase activity-independent apoptosis.

Fig. 1. MLKL-mediated necroptosis causes skin inflammation in FADD^E-KO mice.

A Representative photographs of FADD^E-KO (n = 7) mouse with unaffected littermate (n = 4) at P7 and of FADD^E-KO Mlkl^−/− (n = 8) mouse with WT littermate (n = 8) at 48–50 weeks. B Graph depicting lesion onset of mice with indicated genotypes. C Representative images from skin sections of FADD^E-KO mouse, FADD^E-KO Mlkl^−/− mouse and littermates stained with H&E (Scale bars = 100 µM, n = 7 for FADD^E-KO, n = 4 for control, n = 5 for FADD^E-KO Mlkl^−/−, n = 4 for control) or immunostained with K10, K14 and K6 (Scale bars = 50 µM, n = 4 for FADD^E-KO, n = 3 for control, n = 5 for FADD^E-KO Mlkl^−/−, n = 4 for control). D Graph depicting epidermal thickness of mice with indicated genotypes. E Graphs depicting relative mRNA expression of the indicated cytokines and ISGs in RNA from whole-skin tissue of mice at P7 of the indicated genotypes measured by qRT-PCR. Each dot represents one mouse. Mean ± SEM are shown. F Representative images from skin sections of Casp8^E-KO Mlkl^AA/WT, Casp8^E-KO Mlkl^AA/AA and healthy control stained with H&E (Scale bar 100 µM, n = 4 for Casp8^E-KO Mlkl^AA/WT, n = 4 for Casp8^E-KO Mlkl^AA/AA, n = 4 for control). G Graphs depicting relative mRNA expression of the indicated cytokines and ISGs in RNA from whole-skin tissue of mice at P7-10 of the indicated genotypes measured by qRT-PCR). Statistical significance was determined using Kruskal–Wallis test (one-sided) in (E) and (G).

Fig. 2. TNFR1 and ZBP1 synergize to drive skin inflammation in FADD^E-KO mice.

A Representative photographs of FADD^E-KO TNFR1^E-KO Zbp1^−/WT (n = 15), FADD^E-KO TNFR1^E-KO Zbp1^−/− (n = 27)  and control  (n = 22) mice. B Graph depicting lesion onset of mice with indicated genotypes. C Representative images from skin sections stained with H&E (Scale bars = 100 µM, n = 9 for FADD^E-KO TNFR1^E-KO Zbp1^−/WT, n = 10 for FADD^E-KO TNFR1^E-KO Zbp1^−/−, n = 7 for control) or immunostained with K10, K14 and K6 (Scale bars = 50 µM, n = 3 for FADD^E-KO TNFR1^E-KO Zbp1^−/WT n = 5 for FADD^E-KO TNFR1^E-KO Zbp1^−/−, n = 6 for control). D Graph depicting epidermal thickness of mice with indicated genotypes. E Model depicting pathology induced by loss of FADD and loss of FADD in combination with loss of TNFR1 in keratinocytes.

Fig. 3. C-terminally truncated ZBP1 induces cell death in vitro.

A Scheme depicting structure of FL-ZBP1 and ZBP1ca. B Cell death measured by Draq7 uptake in iMEFs stimulated with combinations of 1 µg doxycycline (dox), emricasan (emri), Nec1s and RIPK3 inhibitor GSK’827. Cell death was normalized to 2 h of Draq5 treatment for each genotype. Representative of at least 3 different experiments. Mean ± SEM are shown. C Immunoblot analysis of protein extracts from iMEFs transduced with doxycycline (Dox)-inducible FL-ZBP1- or ZBP1ca-expressing vectors or stimulated with IFNα. Representative of 3 different experiments. D Anti-Flag immunoprecipitates in iMEFs expressing dox-induced Flag-tagged FL-ZBP1 or Flag-tagged ZBP1ca stimulated with dox. Representative of 5 different experiments. E Immunoblot analysis of protein extracts from iMEFs transduced with Dox-inducible ZBP1ca-, FL-ZBP1 or ZBP1ca mZα-expressing vectors after treatment with increasing Dox concentrations. Representative of 3 different experiments. F Representative images of iMEFs expressing the indicated ZBP1 constructs after overnight doxycycline (+) treatment immunostained for ZBP1. G Cell death measured by Draq7 uptake in iMEFs stimulated with 8 µg dox. Representative of 3 different experiments. Mean ± SEM are shown. H Relative mRNA expression of RNA from iMEFs expressing FL-ZBP1 or ZBP1ca after indicated times of dox treatment. Graph shows mean ± SEM of three different experiments, the same LPS + TNF treated control RNA was used for all experiments.

Fig. 4. ZBP1ca expression in keratinocytes causes skin inflammation.

A Targeting scheme for generation of R26^LSL.ZBP1ca mouse strain using CRISPR Cas9. B Graph depicting lesion onset of mice with indicated genotypes. C Representative photographs of ZBP1ca^E-het mouse (n = 39) and WT littermate (n = 17) at P10. D Immunoblot analysis of whole skin protein lysates from ZBP1ca^E-het (n = 4) and littermate controls (n = 4). Mice were analyzed at P10–12. E Representative images from abdominal and tail skin sections of ZBP1ca^E-het mice and littermates at P10–12 stained with H&E (Scale bars = 100 µM, n = 11 for ZBP1ca^E-het, n = 5 for control), CC3⁺ (n = 5 for ZBP1ca^E-het, n = 2 for R26^LSL.ZBP1ca), TUNEL (n = 4 for ZBP1ca^E-het, n = 2 for R26^LSL.ZBP1ca), or immunostained for ZBP1 (Scale bars = 50 µM, n = 6 for ZBP1ca^E-het, n = 2 for control) or K10, K14 and K6 (Scale bars = 50 µM, n = 9 for ZBP1ca^E-het, n = 11 for control). F Graph depicting epidermal thickness of mice with indicated genotypes at P10–12. Mean + SEM are shown. Each dot represents one mouse. G Graph showing the amount of CC3⁺ cells in abdominal and tail skin of mice with indicated genotypes. Mean ± SEM are shown. Each dot represents one mouse. H Graphs depicting relative mRNA expression of the indicated cytokines and ISGs in RNA from whole-skin tissue of P2 and of P10–12 mice of the indicated genotypes measured by qRT-PCR. Each dot represents one mouse. Mean ± SEM are shown. Statistical significance was determined using Kruskal–Wallis test (one-sided) in (F–H).

Fig. 5. RIPK3 RHIM-dependent signaling promotes skin inflammation in ZBP1ca^E-het mice.

A Graph depicting lesion-free survival of ZBP1ca^E-het, ZBP1ca^E-het Ripk3^mR/mR and healthy littermates. B Graph depicting survival curve of ZBP1ca^E-het, ZBP1ca^E-het Ripk3^mR/mR and healthy littermates. C Graph depicting epidermal thickness of mice with indicated genotypes at P10–12. Mean + SEM are shown. One dot represents one mouse. Data from the same ZBP1ca^E-het mice are included in all Figures for comparison. D Representative images from abdominal and tail skin sections of ZBP1ca^E-het Ripk3^mR/mR, ZBP1ca^E-het and control mice stained with H&E (Scale bars = 100 µM, n = 5 for ZBP1ca^E-het Ripk3^mR/mR, n = 11 for ZBP1ca^E-het, n = 3 for control), CC3⁺ (n = 5 for ZBP1ca^E-het, n = 5 for ZBP1ca^E-het Ripk3^mR/mR, n = 3 for control) or immunostained with K10, K14 and K6 (Scale bars = 50 µM, n = 5 for ZBP1ca^E-het Ripk3^mR/mR, n = 11 for ZBP1ca^E-het, n = 3 for control) at P10–12. E Graph showing the amount of CC3⁺ cells in belly and tail skin of mice with indicated genotypes. Mean ± SEM are shown. Each dot represents one mouse. Data from the same ZBP1ca^E-het mice are included in all Figures for comparison. F Graphs depicting relative mRNA expression of the indicated cytokines and ISGs in RNA from whole-skin tissue of P10–12 mice of the indicated genotypes measured by qRT-PCR. Each dot represents one mouse. Mean ± SEM are shown. Data from the same ZBP1ca^E-het mice are included in all Figures for comparison. Statistical significance was determined using Kruskal–Wallis test (one-sided) in (C, E, F).

Fig. 6. MLKL-dependent necroptosis and to a lesser extent caspase-8-dependent apoptosis cause skin inflammation in ZBP1ca^E-het mice.

A Graph depicting lesion-free survival of ZBP1ca^E-het, ZBP1ca^E-het Mlkl^AA/AA, ZBP1ca^E-het Mlkl^AA/AA Casp8^E-KO and control mice. B Graph depicting survival curve of ZBP1ca^E-het, ZBP1ca^E-het Mlkl^AA/AA and ZBP1ca^E-het Mlkl^AA/AA Casp8^E-KO mice with non-affected littermates. C Representative images from abdominal skin sections of ZBP1ca^E-het, ZBP1ca^E-het Mlkl^AA/AA and ZBP1ca^E-het Mlkl^AA/AA Casp8^E-KO mice and control mice stained with H&E (Scale bars = 100 µM, n = 5 for ZBP1ca^E-het Mlkl^AA/AA, n = 5 for ZBP1ca^E-het Mlkl^AA/AACasp8^E-KO, n = 11 for ZBP1ca^E-het, n = 8 for control), CC3⁺ (n = 5 for ZBP1ca^E-het, n = 4 for ZBP1ca^E-het Mlkl^AA/AA, n = 5 for ZBP1ca^E-het Mlkl^AA/AA/ Casp8^E-KO, n = 5 for control) or immunostained with K10, K14 and K6 (Scale bars = 50 µM, n = 4 for ZBP1ca^E-het Mlkl^AA/AA, n = 5 for ZBP1ca^E-het Mlkl^AA/AA Casp8^E-KO, n = 11 for ZBP1ca^E-het, n = 5 for control) at P10–12. D Graph depicting epidermal thickness of mice with indicated genotypes at P10–12. Mean + SEM are shown. Each dot represents one mouse. Data from the same ZBP1ca^E-het mice are included in all Figures for comparison. E Representative images from tail skin sections of ZBP1ca^E-het, ZBP1ca^E-het Mlkl^AA/AA and ZBP1ca^E-het Mlkl^AA/AA Casp8^E-KO mice and control mice stained with H&E (Scale bars = 100 µM, n = 5 for ZBP1ca^E-het Mlkl^AA/AA, n = 5 for ZBP1ca^E-het Mlkl^AA/AA Casp8^E-KO, n = 11 for ZBP1ca^E-het, n = 8 for control), CC3⁺ (n = 5 for ZBP1ca^E-het, n = 4 for ZBP1ca^E-het Mlkl^AA/AA, n = 5 for ZBP1ca^E-het Mlkl^AA/AA Casp8^E-KO, n = 5 for control) or immunostained with K10, K14 and K6 (Scale bars = 50 µM, n = 4 for ZBP1ca^E-het Mlkl^AA/AA, n = 5 for ZBP1ca^E-het Mlkl^AA/AA Casp8^E-KO, n = 11 for ZBP1ca^E-het, n = 5 for control) at P10–12. F Graph showing the amount of CC3⁺ cells in abdominal and tail skin of mice with indicated genotypes. Mean ± SEM are shown. Each dot represents one mouse. Data from the same ZBP1ca^E-het mice are included in all Figures for comparison. G Graphs depicting relative mRNA expression of the indicated ISGs and cytokines in RNA from whole-skin tissue of P10–12 mice of the indicated genotypes measured by qRT-PCR. Each dot represents one mouse. Mean ± SEM are shown. Data from the same ZBP1ca^E-het and ZBP1ca^E-het Mlkl^AA/AA mice are included in all Figures for comparison. Statistical significance was determined using Kruskal–Wallis test (one-sided) in (D, F, G).

Fig. 7. RIPK1 RHIM-dependent signaling causes MLKL-independent skin inflammation in ZBP1ca^E-het mice.

A Graph depicting lesion-free survival of ZBP1ca^E-het, ZBP1ca^E-het Mlkl^AA/AA and ZBP1ca^E-het Mlkl^AA/AA Ripk1^mR/mR mice and non-affected littermates. B Graph depicting survival curve of ZBP1ca^E-het, ZBP1ca^E-het Mlkl^AA/AA and ZBP1ca^E-het Mlkl^AA/AA Ripk1^mR/mR mice with littermate controls. C Representative images from skin sections of ZBP1ca^E-het and ZBP1ca^E-het Mlkl^AA/AA Ripk1^mR/mR mice and control mice stained with H&E (Scale bars = 100 µM, n = 5 for ZBP1ca^E-het Mlkl^AA/AA Ripk1^mR/mR, n = 11 for ZBP1ca^E-het, n = 5 for control), CC3⁺ (Scale bars = 100 µM, n = 5 for ZBP1ca^E-het Mlkl^AA/AA Ripk1^mR/mR, n = 5 for ZBP1ca^E-het, n = 5 for control), or immunostained with K10, K14 and K6 (Scale bars = 50 µM, n = 5 for ZBP1ca^E-het Mlkl^AA/AA Ripk1^mR/mR, n = 8 for ZBP1ca^E-het, n = 5 for control) at P10–12. D Graph depicting epidermal thickness of mice with indicated genotypes at P10–12. Mean ± SEM are shown. Each dot represents one mouse. Data from the same ZBP1ca^E-het and ZBP1ca^E-het Mlkl^AA/AA mice are included in all Figures for comparison. E Graph showing the amount of CC3⁺ cells in abdominal and tail skin of mice with indicated genotypes. Mean ± SEM are shown. Each dot represents on mouse. Data from the same ZBP1ca^E-het and ZBP1ca^E-het Mlkl^AA/AA mice are included in all Figures for comparison. F Graphs depicting relative mRNA expression of the indicated cytokines in RNA from whole-skin tissue of P10–12 mice of the indicated genotypes measured by qRT-PCR. Each dot represents one mouse. Mean ± SEM are shown. Data from the same ZBP1ca^E-het and ZBP1ca^E-het Mlkl^AA/AA mice are included in all Figures for comparison. Statistical significance was determined using Kruskal–Wallis test (one-sided) in (D–F).

Fig. 8. RIPK1 causes MLKL-independent skin inflammation in ZBP1ca^E-het mice independently of its kinase activity.

A Graph depicting lesion-free survival of ZBP1ca^E-het, ZBP1ca Mlkl^AA/AA, ZBP1ca^E-het Mlkl^AA/AA Ripk1^D138N/D138N mice and non-affected littermates. B Graph depicting survival curve of ZBP1ca^E-het, ZBP1ca Mlkl^AA/AA, ZBP1ca^E-het Mlkl^AA/AA Ripk1^D138N/D138N mice and non-affected littermates. C Representative images from skin sections of ZBP1ca^E-het and ZBP1ca^E-het Mlkl^AA/AARipk1^D138N/D138N mice and control mice stained with H&E (Scale bars = 100 µM, n = 5 for ZBP1ca^E-het Mlkl^AA/AARipk1^D138N/D138N, n = 11 for ZBP1ca^E-het, n = 5 for control), CC3⁺ (n = 5 for ZBP1ca^E-het Mlkl^AA/AARipk1^D138N/D138N, n = 5 for ZBP1ca^E-het, n = 5 for control), or immunostained with K10, K14 and K6 (Scale bars = 50 µM, n = 5 for ZBP1ca^E-het Mlkl^AA/AA Ripk1^D138N/D138N, n = 8 for ZBP1ca^E-het, n = 5 for control) at P10–12. Data from the same ZBP1ca^E-het and ZBP1ca^E-het Mlkl^AA/AA mice are included in all Figures for comparison. D Graph depicting epidermal thickness of mice with indicated genotypes at P10–12. Mean ± SEM are shown. Each dot represents one mouse. Data from the same ZBP1ca^E-het and ZBP1ca^E-het Mlkl^AA/AA mice are included in all Figures for comparison. E Graph showing the amount of CC3⁺ cells in abdominal and tail skin of mice with indicated genotypes. Mean ± SEM are shown. Each dot represents one mouse. F Graphs depicting relative mRNA expression of the indicated ISGs and cytokines in RNA from whole-skin tissue of P10–12 mice of the indicated genotypes measured by qRT-PCR. Each dot represents one mouse. Mean ± SEM are shown. Data from the same ZBP1ca^E-het and ZBP1ca^E-het Mlkl^AA/AA mice are included in all Figures for comparison. Statistical significance was determined using Kruskal–Wallis test (one-sided) in (D–F).