IKKε and TBK1 prevent RIPK1 dependent and independent inflammation

Abstract

TBK1 and IKKε regulate multiple cellular processes including anti-viral type-I interferon responses, metabolism and TNF receptor signaling. However, the relative contributions and potentially redundant functions of IKKε and TBK1 in cell death, inflammation and tissue homeostasis remain poorly understood. Here we show that IKKε compensates for the loss of TBK1 kinase activity to prevent RIPK1-dependent and -independent inflammation in mice. Combined inhibition of IKKε and TBK1 kinase activities caused embryonic lethality that was rescued by heterozygous expression of kinase-inactive RIPK1. Adult mice expressing kinase-inactive versions of IKKε and TBK1 developed systemic inflammation that was induced by both RIPK1-dependent and -independent mechanisms. Combined inhibition of IKKε and TBK1 kinase activities in myeloid cells induced RIPK1-dependent cell death and systemic inflammation mediated by IL-1 family cytokines. Tissue-specific studies showed that IKKε and TBK1 were required to prevent cell death and inflammation in the intestine but were dispensable for liver and skin homeostasis. Together, these findings revealed that IKKε and TBK1 exhibit tissue-specific functions that are important to prevent cell death and inflammation and maintain tissue homeostasis.

Fig. 1. Combined deficiency or kinase inhibition of TBK1 and IKKε cause RIPK1-dependent embryonic lethality and transient alopecia.

a, b Generation of mice with Tbk1^D135N/D135N a, Ikke^K38A/K38A and Ikke^-/- b alleles using CRISPR/Cas9-mediated genome editing. Sanger DNA sequencing of genomic DNA confirmed the generation of the correctly targeted alleles. c, d Expected (E) and observed (O) numbers of weaned offspring with the indicated genotypes from intercrosses of parents with the indicated genotypes. e Immunoblot analysis with the indicated antibodies of whole cell lysates from BMDMs from mice with the indicated genotypes stimulated for 1 hour with 100 ng/ml LPS (top), and ELISA analysis of IFN-β levels in the supernatant of BMDMs from indicated genotypes for 24 h with 100 ng/ml LPS (bottom). Data shown are representative of two independent experiments. f Representative pictures of mice with the indicated age and genotype. g Occurrence of alopecia phenotype in mice with the indicated genotype at pre-weaning age. Source data for e is provided as a source data file.

Fig. 2. Combined deficiency or kinase inhibition of TBK1 and IKKε cause RIPK1-dependent and -independent systemic inflammation.

a, b Graphs showing spleen to body weight ratio a and peripheral blood analysis assayed by differential hematology analyzer b of 8-13 week-old mice with the indicated genotypes. c, d Graphs showing flow cytometry analysis of CD11b + CD115+ (monocytes) and CD11b + Ly6G+ (neutrophils) in bone marrow c and spleen d of 8–13 week-old mice with the indicated genotypes. e Graphs showing ratio of CD69+ (activated) to CD62L+ (naive) T cells amongst live CD45 + CD3+ splenocytes of 8–13 weeks old mice week-old mice with the indicated genotypes. Each dot represents one mouse. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, ^∗∗∗∗p < 0.0001 (one-way ANOVA t-test with post-hoc multiple test). Violin plots show the median of biological replicates and interquartile range of data. Source data for a–e are provided as a source data file.

Fig. 3. Combined inhibition of TBK1 and IKKε kinase activities specifically in myeloid cells causes RIPK1 kinase activity-dependent systemic inflammation.

a Schematic depicting the breeding strategy used to generate mice with combined inhibition of TBK1 and IKKε kinase activities in myeloid cells. Created with BioRender.com. b, c Graph showing spleen to body weight ratio b and representative pictures of spleens c of 8–13 week-old mice with the indicated genotypes. d Graphs depicting peripheral blood analysis of mice with the indicated genotypes, assayed by differential hematology analyzer. e, f Graphs showing cell counts and percentage of CD11b + Ly6G+ (neutrophils) e, as well as CD3 + CD4+ and CD3 + CD8+ cells f among live and CD45+ splenocytes of 8–13 week-old mice with the indicated genotypes. g Graphs showing the ratio of CD69+ (activated) to CD62L+ (naive) T cells amongst live CD45 + CD3+ splenocytes of 8-13 week-old mice with indicated genotypes. Each dot represents one mouse. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, ^∗∗∗∗p < 0.0001 (one-way ANOVA t-test with post-hoc multiple test). Violin plots show the median of biological replicates and interquartile range of data. Source data for b and d–g are provided as a source data file.

Fig. 4. Combined systemic inhibition of TBK1 and IKKε kinase activities causes RIPK1-kinase activity dependent liver damage and inflammation.

a Representative images of liver sections from 8-13 week-old mice with the indicated genotypes immunostained for CD45 and cleaved caspase 8 or stained with hematoxylin and eosin (H&E). Scale bars, 1 mm (black) and 100 μm (green). b, c Serum ALT, ALP and AST levels of 8-13-week-old mice with the indicated genotypes. Each dot represents one mouse. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, ^∗∗∗∗p < 0.0001. (one-way ANOVA t-test with post-hoc multiple test). Violin plots show the median of biological replicates and interquartile range of data. Source data for b, c are provided as a source data file.

Fig. 5. Combined inhibition of TBK1 and IKKε kinase activities specifically in myeloid cells, but not in liver parenchymal cells, causes RIPK1-dependent liver inflammation.

a Representative images of liver sections from 8 to 13 week-old mice with the indicated genotypes immunostained for CD45 and cleaved caspase 8 or stained with hematoxylin and eosin (H&E). Scale bars, 1 mm (black) and 100 μm (green). b, c Serum ALT, ALP, and AST levels of 8-13-week-old mice with the indicated genotypes. Each dot represents one mouse. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, ^∗∗∗∗p < 0.0001. (one-way ANOVA t-test with post-hoc multiple test). Violin plots show the median of biological replicates and interquartile range of data. Source data for b and c are provided as a source data file.

Fig. 6. Combined systemic inhibition of TBK1 and IKKε causes intestinal inflammation.

a Representative images of liver sections from 8 to 13 week-old mice with the indicated genotypes immunostained for CD45 and cleaved caspase 3 or stained with hematoxylin and eosin (H&E). Scale bars, 1 mm (black) and 100μm (green). b, c Graphs showing histological colitis and ileitis scores b and cleaved caspase-3 containing crypt percentage among 100 randomly counted crypts in 8-13 weeks-old mice of the indicated genotypes. Each dot represents one mouse. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, ^∗∗∗∗p < 0.0001. (one-way ANOVA t-test with post-hoc multiple test). Violin plots show the median of biological replicates and interquartile range of data. Source data for b, c are provided as a source data file.

Fig. 7. Combined inhibition of TBK1 and IKKε specifically in myeloid cells causes intestinal inflammation.

a Representative images of liver sections from 8-13 week-old mice with the indicated genotypes immunostained for CD45 and cleaved caspase 3 or stained with hematoxylin and eosin (H&E). Scale bars, 1 mm (black) and 100 μm (green). b, c Graphs showing histological colitis and ileitis scores b and cleaved caspase-3 containing crypt percentage among 100 randomly counted crypts in 8–13 weeks-old mice of the indicated genotypes. Each dot represents one mouse. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, ^∗∗∗∗p < 0.0001. (one-way ANOVA t-test with post-hoc multiple test). Violin plots show the median of biological replicates and interquartile range of data. Source data for b, c are provided as a source data file.

Fig. 8. Combined inhibition of TBK1 and IKKε kinase activities specifically in intestinal epithelial cells causes RIPK1-dependent cell death and intestinal inflammation.

a Representative images of colon and small intestine sections of 5-6 week-old mice with the indicated genotypes immunostained with cleaved caspase 3 or stained with Alcian Blue/PAS. Scale bars, 100 μm. b Graphs showing relative body weight of 5-6 week-old mice with the indicated genotypes. c Graphs displaying the percentage of crypts that have cleaved caspase 3 signal among 100 randomly counted crypts. d Graphs showing relative histological colitis and ileitis scores of 5–6 week-old mice with the indicated genotypes. Each dot represents one mouse. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, ^∗∗∗∗p < 0.0001 (one-way ANOVA t-test with post-hoc multiple test). Violin plots show the median of biological replicates and interquartile range of data. Source data for b–d are provided as a source data file.

Fig. 9. Pharmacological inhibition of TBK1 and IKKε in macrophages causes RIPK1-kinase activity-dependent cell death and IL-1β release.

a, b Graphs showing IL-1β levels in supernatants and Incucyte cell death analysis of BMDMs from wild-type mice stimulated with the indicated amounts of BX795 or MRT67307 in combination with the indicated stimuli, 20μM Necrostatin-s (Nec1s) or 10 μM GSK2982772. IL-1β levels were assessed after 24 a or 20 h b after stimulation. c Graphs showing IL-1β levels in supernatants and Incucyte cell death analysis of BMDMs from mice with the indicated genotypes stimulated with the indicated amounts of MRT67307 for 20 hours. d Graphs showing Incucyte cell death analysis of BMDMs from Ripk1^wt/wt and Ripk1^D138N/D138N mice treated with indicated concentrations of MRT67307. e, f Graphs showing IL-1β levels in supernatants e and Incucyte cell death analysis f of BMDMs from mice with the indicated genotypes stimulated for 24 hours with the indicated amount of BX795 and MRT67307 in combination with the indicated stimuli, 20 μM Nec1s or 10 μM GSK2982772. Results shown were obtained from one experiment in which three independent isolations of BMDMs for each genotype (n = 3 biological replicates) were included. Data are the mean of biological replicates with error bars giving SD. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, ^∗∗∗∗p < 0.0001 (two-way ANOVA t-test with post-hoc multiple test). Source data for a–f are provided as a source data file.

Fig. 10. Combined deficiency in IL-1R1, IL-18R1, and IL-33R suppresses systemic inflammation caused by myeloid cell-specific inhibition of TBK1 and IKKε kinase activity.

a Graphs showing spleen to body weight ratio (left panel), peripheral blood analysis assayed by differential hematology analyzer (middle panel), and ratio of CD69+ (activated) to CD62L+ (naïve) T cells amongst live CD45 + CD3+ cells, as well as the percentage of CD11b + Ly6G+ (neutrophils) amongst live CD45+ cells (right panel) in spleens of 8-13 week-old mice with the indicated genotypes. b Representative images of ileum and colon sections from 8 to 13 week-old mice with indicated genotypes immunostained for CD45 and cleaved caspase-3. Scale bars, 100 μm. c Graphs showing histological colitis and ileitis scores from 8 to 13 week-old mice with indicated genotypes. d Graphs displaying the percentage of crypts that have cleaved caspase 3 signal among 100 randomly counted crypts. Each dot represents one mouse. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, ^∗∗∗∗p < 0.0001 (one-way ANOVA t-test with post-hoc multiple test). Violin plots show the median of biological replicates and interquartile range of data. Source data for a and c, d are provided as a source data file.