Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization (2022)
Figure 9

Figure 9: Elp3 expression in myeloid cells promotes Wnt-driven tumor initiation in the intestine.

A and B Elp3 deficiency in myeloid cells prolongs survival of Apc+/Min mice (A) and reduces the number of adenomas in all parts of the intestine (B). A Kaplan-Meyer curve was established with the indicated genotypes (A). The number of intestinal tumors in the indicated parts of the intestine was quantified in both Apc+/Min Elp3Control and Apc+/Min Elp3∆Mye mice (B) (n = 10 mice per genotype; mean +/- SD, Student T test, ***= p< 0.001, **= p< 0.01, *= p< 0.05). Representative H&E analyses of intestinal crypts from the indicated genotypes are illustrated.

C Elp3 deficiency in myeloid cells decreases the spleen weight of Apc+/Min mice (n = 10 mice per genotype; mean +/- SD, Student T test, **= p< 0.01). Representative pictures of the spleen from the indicated genotypes.

D Elp3 expression in myeloid cells maintains the pool of tumor-associated macrophages (TAMs). The number of TAMs, defined as CD301+/F4/80+ cells among F4/80+ cells was quantified in intestinal crypts from both Apc+/Min Elp3Control and Apc+/Min Elp3∆Mye mice (n = 5 mice per genotype; mean +/- SD, Student T test, *= p< 0.05). Representative immunofluorescence analyses are illustrated.

Expanded Figure 1: Expression of Elongator subunits and Ctu1/2 by M1 polarization signals. A and B M1 polarization signals downregulate the expression of tRNA-modifying enzymes at both mRNA (A) and protein (B) levels. Peritoneal macrophages were untreated or stimulated with IFNγ (50 ng/ml), LPS (100 ng/ml) or with both IFNγ and LPS ex-vivo for the indicated periods of time and mRNA levels of the indicated candidates were assessed in all experimental conditions by Real-Time PCR (A). 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 (n = 3 mice; mean +/- SD, Student T test, * = p<0.05; **= p<0.01; ***= p< 0.001). Protein levels (B) were assessed by western blot analyses using the indicated antibodies.

C BMDMs and intestinal epithelial cells (IECs) lacking Elp3 show decreased thiolated tRNA levels. Northern blots were carried out with total RNAs extracted from BMBMs of Elp3Control and Elp3∆Mye mice or from IECs of Elp3Control and Elp3∆IEC mice, using the indicated probes. Values are from three independent experiments (mean +/- SD, **** = p <0.0001, Welsch one-way Anova test).

D Elp3 expression in myeloid cells is dispensable in the architecture of intestinal crypts. Colons from both Elp3Control and Elp3∆Mye mice were subjected to IHC analyses.

E Colon length is not altered upon Elp3 deficiency in myeloid cells. Data at day 6 from 8 mice per group are illustrated (mean +/- SD, Student T test, non significant).

F mRNA levels of pro-inflammatory cytokines in peritoneal macrophages lacking Elp3 do not change in unstimulated mice. mRNAs of the listed pro-inflammatory cytokines in peritoneal macrophage from Elp3Control or Elp3∆Mye mice were assessed by Real Time PCR. mRNA levels in Elp3Control mice were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels (n = 5 mice; mean +/- SD, non significanr).

Expanded Figure 2: Ctu2 promotes IL-4-dependent mTORC2 activation and M2 macrophage polarization.

A Elp1/3 and Ctu1/2 expression are induced by M2 polarization signals. Peritoneal macrophages were untreated or stimulated with IL-4 (20 ng/ml), IL-13 (20 ng/ml) or with both IL-4 and IL-13 ex-vivo for the indicated periods of time and mRNA levels of the indicated candidates were assessed in all experimental conditions by Real-Time PCR. 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 (n = 3 mice; mean +/- SD, Student T test, * = p<0.05; **= p<0.01; ***= p< 0.001).

B IL-4-dependent mTORC2 activation relies on Ctu2. Control and Ctu2-depleted bone marrow-derived macrophages (BMDMs) were treated or not with IL-4 (10 ng/ml) and the resulting cell extracts were subjected to western blot (WB) analyses.

C Ctu2 deficiency impairs M2 macrophage polarization. Control and Ctu2-depleted BMDMs were treated or not with IL-4 (10 ng/ml) for 24 hours and the resulting mRNAs of the listed M2 markers were assessed by Real Time PCR. mRNA levels in control BMDMs were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels (n = 3 mice) (mean +/- SD, Student T test, *= p< 0.05, **= p< 0.01, ***= p< 0.001).

Expanded Figure 3: Metabolic reprogramming by IL-4 relies on Elp3. Metabolic reprogramming by IL-4 relies on Elp3. Peritoneal macrophages from Elp3Control and Elp3∆Mye mice (n = 3 per genotype) were treated with IL-4 for 24 hours ex-vivo and the resulting extracts were subjected to Mass Spec analyses to extensively quantify metabolites. At the bottom, defective TCA cycle upon Elp3 deficiency in IL-4-treated macrophages. Red arrows identify TCA metabolites whose levels change upon Elp3 deficiency.

Expanded Figure 4: Elp3 promotes Ric8b expression.

A Elp3 does not regulate Ric8b mRNA levels. mRNAs of BMDMs from the indicated genotypes and stimulated ex-vivo with IL-4 or with PBS (control) were subjected to Real-Time PCRs. mRNA levels of Ric8b in BMDMs treated with PBS from Elp3Controlmice were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels (n = 3 mice per genotype (mean +/- SD).

B Ric8b promotes LPS-dependent mTORC2 activation. Control or Ric8b-depleted BMDMs were treated or not with IFNγ (50 ng/ml) and LPS (100 ng/ml) for the indicated periods of time and the resulting cell extracts were subjected to WB analyses.

C Increased aggregate formation by IL-4 in macrophages. Flow cytometry of aggresomes with extracts of BMDMs from Elp3Control and Elp3∆Mye mice treated with IL-4 (10 ng/ml) for 24 hours are illustrated. Experiments were conducted in duplicates.

D Visualization of protein aggregates. Coomassie blue staining of aggregate samples from Elp3Control and Elp3∆Mye BMDMs treated with IL-4 or PBS (control) (n= 8 mice).

E Identification of a proteomic signature of candidates whose expression is increased upon IL-4 stimulation and in BMDMs lacking Elp3 (Venn diagram).

F Identification of proteins enriched in aggregates in macrophages lacking Elp3. Dot plot of protein aggregates from IL-4-treated BMDMs cells lacking or not Elp3 are shown. Genes enriched in LysAAA, GlnCAA, and GluGAA codons are shown in blue. Mrpls are shown in Red.

G The expression of selected Mrpl proteins is controlled by Elp3 in macrophages. Peritoneal macrophages from Elp3Control and Elp3∆Mye mice were treated or not with IL-4/IL-13 (10 ng/ml) for the indicated periods of time and cell extracts from resulting cells were subjected to WBs using the indicated antibodies.

H Elp3 does not regulate mRNA levels of Mrpl proteins. PMs from the indicated genotypes were stimulated or not with IL-4/IL-13 (10 ng/ml) for 24 hours and mRNA levels of the indicated candidates were quantified by Real-Time PCR. mRNA levels of all candidates in untreated cells from Elp3Control mice were set to 1 and levels in other experimental conditions were relative to that after normalization with Gapdh mRNA levels (n = 3 mice, mean +/- SD, Student T test, non significant).

I Elp3 controls Mt-co3 expression in macrophages. Protein extracts from PMs of the indicated genotypes treated or not with IL-4/IL-13 (10 ng/ml) for the indicated periods of time were subjected to WB analyses.

Expanded Figure 5: Elp3 deficiency in peritoneal macrophages does not stabilize Atf4.

A Peritoneal macrophages from the indicated genotypes were stimulated or not with IL-4/IL-13 (10 ng/ml) for the indicated periods of time and the resulting extracts were subjected to WB analyses.

B Elp3 deficiency does not potentiates Atf4-driven transcription in myeloid cells. Peritoneal macrophages from the indicated genotypes were stimulated or not with IL-4 (10 ng/ml) for 24 hours and mRNA levels of the indicated Atf4 target genes were quantified by Real-Time PCR. mRNA levels of all candidates in untreated cells 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, non significant).