Divergent roles of RIPK3 and MLKL in high-fat diet–induced obesity and MAFLD in mice

Figures

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  Figure S1.. NLRP3 inflammasome is required for palmitate-induced IL-1β activation in LPS-primed macrophages. (A, B, C, D) WT, Nlrp3−/−, and Casp1−/− BMDMs were pretreated with and without LPS (50 ng/ml) for 3 h and treated with 300–600 μM palmitate conjugated to BSA (PA-BSA) or BSA alone (equivalent to 600 μM BSA amount) for a further 18–20 h. (A) IL-1β and (C) TNF levels were measured in cell supernatants by ELISA. Data shown are the mean ± SD of n = 3 independent biological experiments with four to five technical replicates averaged per experiment. (B) Cell lysates and supernatants were analyzed by immunoblot for the indicated proteins. The results shown are representative of two independent experiments. (D) Cell viability was assessed by PI incorporation and flow cytometric analysis. Data shown are the mean ± SD of n = 3 independent biological experiments with four to five technical replicates averaged per experiment. One-way ANOVA followed by Tukey’s multiple comparison test, *P < 0.05, ****P < 0.0001. (E, F, G) WT, Nlrp3−/−, and Casp1−/− BMDMs were primed with LPS (50 ng/ml) for 3 h and treated with nigericin (10 μM) for ∼45 min. (E) Cell lysates and supernatants were analyzed by…

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  Figure 1.. NLRP3 inflammasome priming, IL-1β activation, and cell death are defective in Ripk3−/−Casp8−/− macrophages in response to LPS and palmitate. (A, B, C, D) WT, Mlkl−/−, Ripk3−/− and Ripk3−/−Casp8−/− BMDMs were primed with or without LPS (50 ng/ml) for 3 h and treated with 600 μM PA-BSA or BSA alone, as indicated, for 18–20 h. (A) Cell lysates and supernatants were analyzed by immunoblot for specified proteins. The results shown are representative of two independent biological experiments. (B) IL-1β and (C) TNF levels were measured in cell supernatants by ELISA. Data shown are the mean ± SEM of n = 3 biological replicates and are representative of at least four independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, ****P < 0.0001. (D) Cell viability was assessed by PI uptake and flow cytometric analysis. Data shown are the mean ± SEM of n = 3 biological replicates and are representative of at least four independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, ****P < 0.0001. Source data are available for this figure.

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  Figure S2.. Defective caspase-8–mediated inflammasome priming reduces palmitate-induced NLRP3 inflammasome activation. (A) WT, Mlkl−/−, Ripk3−/−, and Ripk3−/−Casp8−/− BMDMs were primed with or without with LPS (50 ng/ml) for 3 h and treated with 600 μM PA-BSA or BSA alone, as indicated, for 18–20 h. Cell lysates were subjected to immunoblot for RIPK3, caspase-8, and MLKL. Results shown are representative of one experiment. (B, C) WT, Mlkl−/−, Ripk3−/−, and Ripk3−/−Casp8−/− BMDMs were primed with LPS (50 ng/ml) for 3 h and treated with nigericin (10 μM) for ∼45 min (B) IL-1β levels were measured in cell supernatants by ELISA. (C) Cell viability was assessed by PI incorporation and flow cytometric analysis. Data shown are the mean ± SEM of n = 3 biological replicates and are representative of at least four independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, **P < 0.01. (D, E, F, G) WT, Mlkl−/−, Ripk3−/−, and Ripk3−/−Casp8−/− BMDMs were primed with or without Pam3Cys (500 ng/ml) for 3 h and treated with 600 μM PA-BSA or BSA alone, as indicated, for 18–20 h. (D) IL-1β and (F) TNF levels were measured in cell supernatants by ELISA. Data are the mean ± SEM of n = 3…

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  Figure 2.. RIPK3 deficiency and myeloid-specific loss of caspase-8 reduce HFD-induced metabolic dysfunction, adipose tissue inflammation, and MAFLD development. (A, B) Body weights were measured weekly in Casp8lox/lox control (WT), Casp8lox/loxRipk3−/−, and Casp8LysMcreRipk3−/− mice fed a (A) normal chow diet (ND) or (B) high-fat diet (HFD) for ∼25 wk. Data shown are the mean ± SEM, n ≥ 11 ND-fed mice per group and n ≥ 13 HFD-fed mice per group pooled from three independent experiments. One-way ANOVA of the AUC, **P < 0.01. (C) End-stage organ weights in HFD-fed Casp8lox/lox control, Casp8lox/loxRipk3−/−, and Casp8LysMcreRipk3−/− mice. Data shown are the mean ± SEM, n ≥ 11 mice per group pooled from three independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, *P < 0.05. Gray boxes in (C) show the mean ± SEM from Casp8lox/lox, Casp8lox/loxRipk3−/−, and Casp8LysMcreRipk3−/− ND-fed mice (Fig S3D) for comparisons. (D, E) Glucose tolerance was measured in (D) ND- and (E) HFD-challenged Casp8lox/lox control, Casp8lox/loxRipk3−/−, and Casp8LysMcreRipk3−/− mice by intraperitoneal glucose tolerance tests (IP-GTT; 1.5 g/kg) at 8 wk. Data shown are the mean ± SEM, n = 3–6…

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  Figure S3.. Mice lacking RIPK3 and caspase-8 in myeloid cells are protected from weight gain and fatty liver upon HFD challenge. (A, B) % Weight gain over time in Casp8lox/lox control (WT), Casp8lox/loxRipk3−/−, and Casp8LysMcreRipk3−/− mice fed a (A) normal chow diet (ND) or a (B) high-fat diet (HFD). Data shown are the mean ± SEM, n ≥ 11 mice per group pooled from three independent experiments. One-way ANOVA of the AUC, *P < 0.05, **P < 0.01, ***P < 0.001. (C) ND and HFD food intake and output per mouse per day were calculated from weekly measurements in Casp8lox/lox control, Casp8lox/loxRipk3−/−, and Casp8LysMcreRipk3−/− mice. Data shown are the mean + SD, n ≥ 8 mice per group from two pooled independent experiments, one-way ANOVA. (D, E, F) End-stage (25 wk of diet) organ weights and (G) organ weights expressed as a % of total body weight in Casp8lox/lox control, Casp8lox/loxRipk3−/−, and Casp8LysMcreRipk3−/− mice. Data shown are the mean ± SEM, n ≥ 11 mice/group pooled from three independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test, *P < 0.05, **P < 0.01. Gray boxes in (F) show the mean ± SEM from Casp8lox/lox control, Casp8lox/loxRipk3−/−, and…

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  Figure S4.. Mice lacking RIPK3 and caspase-8 in myeloid cells are modestly protected from HFD-induced metabolic dysfunction. (A, B, C) Intraperitoneal glucose tolerance (GTT, 1.5 g/kg) and (D) insulin tolerance tests (ITT, 0.75 U/kg) were performed, as indicated, in ND- and HFD-fed Casp8lox/lox control, Casp8lox/loxRipk3−/−, and Casp8LysMcreRipk3−/− mice, and blood glucose levels were monitored over time. Data shown are the mean ± SEM, n = 5–6 mice per group and are representative of one of two to three experiments. For ITT, blood glucose changes are normalized to baseline measurements. One-way ANOVA of the AUC, *P < 0.05. (E, F, G, H, I, J) Fasting serum, (E) glucose, (F) insulin, (G) triglyceride, (H) cholesterol, (I) ALT, and (J) AST levels in Casp8lox/lox control, Casp8lox/loxRipk3−/−, and Casp8LysMcreRipk3−/− ND- and HFD-fed mice after 16–18 (diamonds) or 25 (triangles/circles) wk. Data shown are the mean ± SEM, n ≥ 9 mice per group pooled from at least three experiments. One-way ANOVA followed by Tukey’s multiple comparison test, *P < 0.05, **P < 0.01. The dotted line shows the mean values of control Casp8lox/lox ND-fed mice extrapolated for comparison.

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  Figure S5.. Loss of RIPK3 and caspase-8 activity contributes to tissue inflammation in HFD-induced obesity. (A) Representative microscopy images of VAT sections from Casp8lox/lox control, Casp8lox/loxRipk3−/−, and Casp8LysMcreRipk3−/− mice fed a ND or HFD for 25 wk stained with H&E (top row; the scale bar is 200 μm) or cleaved caspase-3 immunohistochemistry (bottom row; the scale bar is 100 μm). (B) Mean adipocyte size as quantified on VAT H&E sections from Casp8lox/lox control, Casp8lox/loxRipk3−/−, and Casp8LysMcreRipk3−/− mice fed a ND. Data shown are the mean ± SEM, n = 6–12 mice pooled from three independent experiments, one-way ANOVA. (C) Flow cytometric gating strategy for analyzing live CD45+ PI− myeloid populations in the VAT of HFD mice after 25 wk. (D) Representative microscopy images of Sirius Red–stained liver tissue sections showing fibrosis in ND- and HFD-fed Casp8lox/lox, Casp8lox/loxRipk3−/−, and Casp8LysMcreRipk3−/− mice. The scale bar is 200 μm. (E) Flow cytometric gating strategy for analyzing live CD45+ PI− myeloid populations in the liver of mice fed a HFD for 25 wk. (F, G) Flow cytometric analysis showing the (F) % of CD45+ leukocytes and (G) proportion of neutrophils…

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  Figure 3.. Mlkl−/− mice display reduced obesity and metabolic dysfunction with aging and with HFD feeding. (A, C) WT and Mlkl−/− mice were fed an (A) ND and (C) HFD for ∼24–25 wk and body weights measured on a weekly basis. Data shown are the mean ± SEM, n ≥ 14 ND-fed mice/group and n ≥ 23 HFD-fed mice/group pooled from three independent experiments. Unpaired, two-tailed t test of the AUC, *P < 0.05, ***P < 0.001. (B, D) End-stage organ weights from (B) ND- and (D) HFD-fed WT and Mlkl−/− mice. Data shown are the mean ± SEM, n ≥ 14 mice/group pooled from three independent experiments. Unpaired, two-tailed t test, ***P < 0.001, ****P < 0.0001. Gray boxes in (D) represent the mean ± SEM from (B) for comparison. (E, F, G, H) Glucose tolerance was assessed via an IP-GTT (1.5 g/kg) at 8 and 17–18 wk in ND- and HFD-fed WT and Mlkl−/− mice. Data shown are the mean ± SEM, (E, F) n = 5–6 ND-fed mice per group and (G, H) n = 8–9 HFD-fed mice per group and are representative of one of 2–3 experiments. Unpaired, two-tailed t test of the AUC, *P < 0.05. (I) Insulin resistance was assessed in HFD-fed WT and Mlkl−/− mice (∼23 wk) during an IP-ITT (0.75 U/kg). Data shown are the mean ± SEM, n = 8–9…

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  Figure S6.. MLKL-deficient mice display reduced obesity with aging and upon HFD feeding. (A, B) % Weight gain over time in WT and Mlkl−/− mice fed a (A) ND or (B) HFD for 25 wk. Data shown are the mean ± SEM, n ≥ 14 ND-fed mice/group and n ≥ 23 HFD-fed mice per group pooled from three independent experiments. Unpaired, two-tailed t test of the AUC, ****P < 0.0001. (C, D) End-stage organ weights and (E) organ weights expressed as a % of total body weight in WT and Mlkl−/− mice fed a ND or HFD for 25 wk. Data shown are the mean ± SD, n = 11–17 mice per group pooled from three independent cohorts. Unpaired, two-tailed t test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Gray boxes in (D) show the mean ± SEM from WT and Mlkl−/− ND-fed mice (C). (F) ND and HFD food intake and output per mouse per day were calculated from weekly measurements in WT and Mlkl−/− mice. Data shown are the mean + SD, n ≥ 9 ND-fed mice per group and n ≥ 20 HFD-fed mice/group pooled from 2 independent experiments. Unpaired, two-tailed t test, ****P < 0.000. (G) ITT (0.75 U/kg) was performed in ND-fed WT and Mlkl−/− mice at 23 wk, and blood glucose was monitored over time. Data are the mean ± SEM, n = 5–6 mice per…

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  Figure 4.. MLKL deficiency reduces adiposity and fatty liver disease in response to HFD challenge. (A) Representative microscopy images of H&E-stained and F4/80-immunostained VAT sections from WT and Mlkl−/− mice fed a ND or HFD for ∼25 wk. Arrows point to crown-like structures. The scale bar is 100 μm. (B) Automated quantification of the mean adipocyte size in VAT on H&E-stained sections (A). Data shown are the mean ± SEM, n ≥ 13 ND-fed mice per group and n ≥ 23 HFD-fed mice per group pooled from three independent experiments. Unpaired, two-tailed t test, **P < 0.01, ***P < 0.001. (C) VAT from HFD-fed WT and Mlkl−/− mice was harvested at ∼25 wk, and the numbers of neutrophils, inflammatory monocytes, and macrophages were quantified by flow cytometric analysis. Data shown are the mean ± SEM, n ≥ 14 mice per group pooled from two independent experiments. Unpaired, two-tailed t test. (D) VAT from HFD-fed WT and Mlkl−/− mice was harvested at ∼25 wk and cultured ex vivo with and without LPS (50 ng/ml) overnight, and IL-1β and TNF were measured in the supernatants by ELISA. Data shown are the mean ± SEM, n ≥ 19 mice per group from pooled from three independent experiments. Unpaired, two-tailed t…

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  Figure S7.. WT mice reconstituted with Mlkl−/− bone marrow develop obesity and metabolic dysfunction. (A) Weight gain and (B) fat mass as a % of total body weight (assessed weekly using an EchoMRI) in WT and Mlkl−/− bone marrow transfer recipients fed a ND or HFD for 23 wk. Data are the mean ± SEM, n = 12 mice per group. Unpaired, two-tailed t test of the AUC. (C) End-stage organ weights were measured in WT and Mlkl−/− bone marrow chimeras fed a ND or HFD. Data are the mean ± SEM, n = 12 mice per group. Unpaired, two-tailed t test. (D) Oral GTT (oral gavage 2 g/kg based on lean body mass) was performed on WT and Mlkl−/− bone marrow chimeras after 10 wk of ND and HFD feeding, and blood glucose levels were monitored over time. Data are the mean ± SEM, n = 12 mice per group. Unpaired, two-tailed t test of the AUC.

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  Figure 5.. Deficiency in MLKL leads to altered gene signatures in the livers of aging and HFD-fed mice. (A, B, C, D) Liver RNA extracted from WT and Mlkl−/− mice on a ND or HFD (n = 3 mice per group) was subjected to 3′ mRNA sequencing. (A) Multidimensional scaling plot and (B) the number of differentially expressed genes up-regulated and down-regulated in WT v Mlkl−/− ND and HFD livers. P ≤ 0.05 and cutoff values logFC ≥ 1 or logFC ≤ −1. (C) Gene ontology (GO) pathways of significant DEGs up-regulated and down-regulated in Mlkl−/− ND livers compared with WT ND livers. P ≤ 0.05 and cutoff values logFC ≥ 1 or logFC ≤ −1. (D) Top 30 GO pathways of significant DEGs down-regulated in Mlkl−/− HFD livers compared with WT HFD livers. P ≤ 0.05 and cutoff values logFC ≤ −1. Source data are available for this figure.