Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization (2022)
Figure 3

Figure 3: Elp3 induction by IL-4 through multiple signaling pathways.

A IL-4 and IL-13 induce the expression of tRNA-modifying enzymes in macrophages. Peritoneal macrophages were untreated (« Control ») or stimulated with IL-4 (10 ng/ml), IL-13 (10 ng/ml) or with both cytokines for 24 hours and cell extracts from the resulting cells were subjected to WB analyses.

B The pool of thiolated tRNAs in macrophages is enhanced by IL-4/IL-13 but decreased by LPS/IFNγ. On the left, Northern blot analysis assessing t:Q(UUG) tRNA thiolation in Thioglycollate-elicited peritoneal macrophages subjected to the indicated treatments for 24 hours. On the right, quantification of t:Q(UUG) tRNA thiolation, calculated as the ration of thiolated over nonthiolated t:Q(UUG), in the indicated experimental conditions.

C Elp3 induction by IL-4/IL-13 occurs through a PI3K-dependent pathway. Peritoneal macrophages were pretreated with DMSO (control vehicle) or with Wortmannin (200 nM) for one hour and subsequently left untreated or stimulated with IL-4 (10 ng/ml)/IL-13 (10 ng/ml) for the indicated periods of time. Cell extracts from the resulting cells were subjected to WB analyses.

D PI3K inhibition does not impact on Elp3 mRNA levels. Peritoneal macrophages were pretreated with DMSO (control vehicle) or with Wortmannin (200 nM) for one hour and subsequently left untreated or stimulated with IL-4 (10 ng/ml) for 24 hours. Elp3 mRNA levels were quantified in all experimental conditions by Real-Time PCR. Elp3 mRNA levels in unstimulated cells were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels. Experiments were conducted in triplicates (mean +/- SD, Student T test, **= p< 0.01).

E Rictor but not Raptor deficiency impairs Elp3 expression. BMDMs were transfected with the indicated siRNAs and the resulting cells were treated or not with IL-4 (10 ng/ml)/IL-13 (10 ng/ml) for 24 hours. Cell extracts were subjected to WB analyses.

F IL-4/IL-13 rely on Stat6 to induce Elp3 expression. Peritoneal macrophages were pretreated with the control vehicle (DMSO) or with the Stat6 pharmacological inhibitor AS1517499 (5 μM) for one hour and subsequently left unstimulated or treated with IL-4 (10 ng/ml)/IL-13 (10 ng/ml) for the indicated periods of time. Cell extracts from the resulting cells were subjected to WB analyses. G Stat6 is recruited on the Elp3 promoter in an IL-4-dependent manner. ChIP assays using an anti-Stat6 antibody were conducted in RAW 264.7 cells to assess Stat6 recruitment on a Stat-DNA binding site found in the Elp3 promoter. IgG antibody was used as a negative result. The histogram shows Stat6 recruitment on the Stat DNA binding site with or without IL-4 (10 ng/ml) treatment for 2 hours. Results obtained with four independent experiments (mean +/- SD, Student T test, *= p< 0.05) are illustrated.

H to J Elp3 deficiency in myeloid cells impairs multiple signaling pathways triggered by IL-4 (H, I and J), IL-13 (H) or by both cytokines (H). Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL-4 (10 ng/ml) (H, I and J), IL-13 (10 ng/ml) (H) or with both cytokines (H) for 8 hours (H) or for the indicated periods of time (I and J) and the resulting cell extracts were subjected to WB analyses.

K Elp3 deficiency in BMDMs impairs the production of M2 markers upon stimulation with IL-4/IL-13. mRNAs of M2 markers were assessed by Real Time PCR. mRNA levels in unstimulated macrophages from Elp3Control mice were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels (n = 4 mice; mean +/- SD, Student T test, *= p< 0.05, ***=p<0.001).

L Elp3 promotes M2 macrophage polarization. Peritoneal macrophages isolated from the indicated mouse genotypes were untreated or stimulated with IL-4/IL-13 (10 ng/ml) for 24 hours and the resulting cell extracts were subjected to WB analyses.