Interleukin-33 regulates metabolic reprogramming of the retinal pigment epithelium in response to immune stressors
Louis M. Scott, Emma E. Vincent, Natalie Hudson, Chris Neal, Nicholas Jones, Ed C. Lavelle, Matthew Campbell, Andrew P. Halestrap, Andrew D. Dick, Sofia Theodoropoulou
Louis M. Scott, Emma E. Vincent, Natalie Hudson, Chris Neal, Nicholas Jones, Ed C. Lavelle, Matthew Campbell, Andrew P. Halestrap, Andrew D. Dick, Sofia Theodoropoulou
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Research Article Metabolism Ophthalmology

Interleukin-33 regulates metabolic reprogramming of the retinal pigment epithelium in response to immune stressors

Abstract

It remains unresolved how retinal pigment epithelial cell metabolism is regulated following immune activation to maintain retinal homeostasis and retinal function. We exposed retinal pigment epithelium (RPE) to several stress signals, particularly Toll-like receptor stimulation, and uncovered an ability of RPE to adapt their metabolic preference on aerobic glycolysis or oxidative glucose metabolism in response to different immune stimuli. We have identified interleukin-33 (IL-33) as a key metabolic checkpoint that antagonizes the Warburg effect to ensure the functional stability of the RPE. The identification of IL-33 as a key regulator of mitochondrial metabolism suggests roles for the cytokine that go beyond its extracellular “alarmin” activities. IL-33 exerts control over mitochondrial respiration in RPE by facilitating oxidative pyruvate catabolism. We have also revealed that in the absence of IL-33, mitochondrial function declined and resultant bioenergetic switching was aligned with altered mitochondrial morphology. Our data not only shed new light on the molecular pathway of activation of mitochondrial respiration in RPE in response to immune stressors but also uncover a potentially novel role of nuclear intrinsic IL-33 as a metabolic checkpoint regulator.

Authors

Louis M. Scott, Emma E. Vincent, Natalie Hudson, Chris Neal, Nicholas Jones, Ed C. Lavelle, Matthew Campbell, Andrew P. Halestrap, Andrew D. Dick, Sofia Theodoropoulou

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Figure 7

Nuclear IL-33 promotes oxidative glucose metabolism.

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Nuclear IL-33 promotes oxidative glucose metabolism. (A and B) ARPE-19 w...
(A and B) ARPE-19 were transfected with either an IL-33 activation plasmid or scrambled gRNA activation plasmid; (A) RNA was extracted, and RT-PCR was used to determine the expression of MPC1 and MPC2 (n = 3); (B) protein was extracted and Western blot analysis was used to determine the expression of MPC1 and MPC2 (n = 3). (C and D) ARPE-19 were transfected with either an IL-33 siRNA or scrambled siRNA; (C) RNA was extracted, and RT-PCR was used to determine the expression of MPC1 and MPC2 (n = 3); (D) protein was extracted and Western blot analysis was used to determine the expression of MPC1 and MPC2 (n = 3). (E) Modified mitochondrial stress test following transfection of ARPE-19 with either an IL-33 activation plasmid or scrambled gRNA activation plasmid; XF injections were oligomycin (1 μM), FCCP (0.5 μM), UK5099 (5 μM), and rotenone/antimycin A (1 μM) (n = 3). (F) Parameter calculated from E (as detailed in Methods) (n = 3). (G) Modified mitochondrial stress test following transfection of ARPE-19 with either an IL-33 siRNA or scrambled siRNA; XF injections were oligomycin (1 μM), FCCP (0.5 μM), UK5099 (5 μM), and rotenone/antimycin A (1 μM) (n = 3). (H) Parameter calculated from G (as detailed in Methods) (n = 3). (I) Uniformly labeled C13-glucose incorporation into ARPE-19 TCA cycle metabolites following transfection with either an IL-33 activation plasmid or a scrambled gRNA activation plasmid control; relative abundance of C13 and C12 including succinate, fumarate, malate, and citrate (n = 3). (J) Uniformly labeled C13-glucose incorporation into ARPE-19 TCA cycle metabolites following transfection with either an IL-33 siRNA or scrambled siRNA control (n = 3). Relative abundance of C13 and C12 including succinate, fumarate, malate, and citrate (n = 3). (K and L) Mass isotopolog distributions (MIDs) of ARPE-19 TCA cycle intermediates (K) M+6 citrate and (L) M+4 malate, following transfection with either an IL-33 activation plasmid or a scrambled gRNA activation plasmid (n = 3). (M) ARPE-19 were transfected with either an IL-33 activation plasmid/scrambled gRNA activation plasmid or an IL-33 siRNA/scrambled siRNA; Western blot analysis was used to determine the phosphorylation status of PDH (n = 3). (N) Citrate M+3/pyruvate M+3 ratio in ARPE-19 transfected with either an IL-33 activation plasmid or scrambled gRNA activation plasmid (n = 3). Data are expressed as means ± SD from at least 3 independent experiments. (E–H) Represent the biological repeats from 3 independent experiments (n = 3); each biological repeat is the mean of 3 technical repeats (3 seahorse wells per experiment). Unpaired Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001.