Inhibition of Aurora kinase A suppresses trained immunity in macrophages.

(A) BMDMs were trained with β-glucan at a dosage of 50 μg/mL in the presence of different concentration of alisertib for 24 h. The viability of BMDMs was measured by CCK8. (B) Supernatant levels of IL-6 (left) and TNF-α (right) in trained BMDMs with alisertib (0.5 μM or 1 μM), followed by restimulation with LPS (20 ng/mL) for 24 h. (C) Relative mRNA expression of Il6 and Tnfα in trained BMDMs with alisertib (0.5 μM or 1 μM), followed by restimulation with LPS (20 ng/mL) for 6 h. (D) Immunoblotting analysis of AurA phosphorylation after the treatment of β-glucan (50 μg/mL) or alisertib (1 μM) for 90 min. (E) Immunoblotting analysis of AurA in BMDMs transfected with siRNA targeting AurA for 48 h. (F) The BMDMs were firstly transfected with siRNAs for 48 hours and then followed by β-glucan training. Supernatant levels of IL-6 and TNF-α were detected by ELISA after 3 days rest and restimulation with LPS. (G) The BMDMs was trained with BMDMs (50 μg/mL) or in combination with alisertib or with AurA knocking down, followed by a rest for 3 days and restimulation with cell culture supernatant from MC38. (H) Graphical outline of in vivo training model (mice n=3 per group). (I) Supernatant levels of IL-6 (left) and TNF-α (right) in trained BMDMs as shown in H; each point in the graph represents an individual mouse. Data are representative of three independent experiments (except in I) and presented as the mean ± SEM. P values were derived from one-way ANOVA test with a Dunnett’s multiple-comparison test (A, F, G, I) or two-way ANOVA with a Tukey’s multiple-comparison test (B, C). Related to Figure 1—figure supplement 1, Figure 1—source data 1-2.

Aurora kinase A inhibition remodels chromatin landscape of inflammatory genes.

(A) Principal component analysis (PCA) of gene peaks in ATAC-seq. (B) GO enrichment analysis of erased peaks by alisertib in trained BMDMs. (C) Representative motifs in the erased (n=15,431) and written (n=19,733) peaks respectively. (D and E) Genome browser views of ATAC-seq signal of representative genes inhibited by alisertib including Cxcl2, Il1a, Tnfα and Il6 (D) and representative genes enhanced by alisertib including Mrc1 and Chil3 (E). (F and G) KEGG enrichment of differentially expressed genes in trained BMDMs rechallenged with LPS; alisertib downregulated genes (F) and upregulated genes (G) were mapped into KEGG respectively. Related to Figure 2—figure supplement 1.

Aurora kinase A inhibition decreases glycolysis and SAM level.

(A and B) Extracellular acidification rate (ECAR) after a glycolysis stress test upon sequential addition of glucose (Gluc, 10 mM), oligomycin (Oligo, 1μM), and 2-deoxyglucose (2-DG, 50 mM), as indicated in BMDMs with different treatment for 24 h (A); basal glycolysis and maximal glycolysis (B). (C, F and G) LC–MS/MS measurements of fumarate (C), serine and SAM (F), SAH and HCY (G) in trained BMDMs treated with vehicle or alisertib for 24 h. (D) BMDMs were trained with β-glucan (50 μg/mL) or combined with alisertib (1μM) for 24 h. The BMDMs were collected for RNA extraction and followed by RNA-seq. The TOP 10 enriched pathways identified by KEGG enrichment analysis of differentially expressed genes (Foldchange >1.2, FDR<0.05) by comparing alisertib inhibited with un-inhibited trained BMDMs. (E) Intracellular level of glutathione in trained BMDMs with vehicles or alisertib for 24 h. The level was normalized to untrained BMDMs. (H) Western blot analysis of GNMT in trained BMDMs treated with vehicles or alisertib for 24 h. β-actin was used as a loading control; * showed the position of GNMT blot. (I) Western blot to detect GNMT protein in wild type BMDMs that were transfected with small interferon RNA targeting GNMT. (J) LC–MS/MS measurements of SAM and SAH in alisertib-inhibited trained BMDMs with knockdown of GNMT. The SAM/SAH ratio is calculated by SAH normalization. P values were derived from unpaired one-tailed way t-test. (K) Supernatant level of IL-6 and TNF-α in trained BMDMs with AurA inhibition by alisertib or by siRNA targeting Aura, together with without GNMT deficiency. Data are representative of three independent experiments and presented as the mean ± SEM. P values were derived from one-way ANOVA test with a Turkey’s multiple-comparison test (B) or with a Dunnett’s multiple-comparison test (C, E-G, K). Related to Figure 3—figure supplement 1, Figure 3—source data 1-2.

Inhibition of Aurora kinase A impairs histone trimethylation at H3K4 and H3K36.

(A) Western blot analysis of histone methylation modifications in trained BMDM treated with vehicles or alisertib. Histone 3 (H3) was used as a loading control. BMDMs were trained with β-glucan (50 μg/mL) or combined with alisertib (1μM) for 24 h, then BMDMs were washed and cultured in fresh medium for 3 days, followed by protein extraction. (B) ChIP-qPCR analysis of H3K4me3 and H3K36me3 enrichment in IL-6 and TNF-α in trained BMDMs treated with vehicles or alisertib for 24 h and rest for 3 days. (C) Western blot analysis of total H3K4me3 and H3k36me3 upon GNMT deficiency in BMDMs. The BMDMs were transfected with siRNA targeting GNMT for 48 h, followed by β-glucan (50 μg/mL) or combination with alisertib (1 μM) treatment for 24 h. Then the BMDMs were washed and cultured in fresh medium for 3 days and the protein was extracted for western blot analysis of H3K4me3 and H3K36me3. Data are representative of three independent experiments and presented as the mean ± SEM. P values were derived from one-way ANOVA test with a Turkey’s multiple-comparison test. Related to Figure 4—figure supplement 1, Figure 4—source data 1-2.

Aurora kinase A regulates GNMT through transcription factor FOXO3.

(A) Protein level of GNMT under FOXO3 deficiency in BMDMs without treatment was detected by western blot; * showed the position of FOXO3 blot. (B and C) Western blot analysis of GNMT downregulation by siFoxo3 in trained β-glucan with AurA inhibition. BMDMs were transfected with smalling interferon RNA targeting FOXO3 for 48 h, followed by β-glucan training and alisertib for 24 h (B); BMDMs were transfected with smalling interferon RNA targeting FOXO3 and AurA for 48 h, followed by β-glucan training for another 24 h (C). (D) Supernatant levels of IL-6 and TNF-α in BMDMs. The cells were treated with β-glucan training and aurora A inhibition in combination with FOXO3 deficiency by siRNA. (E) Western blot analysis of phosphorylation level of FOXO3 at ser 315 in BMDMs treated with β-glucan (50 μg/mL) or combined with alisertib (1 μM) for 12 h. (F) Immunofluorescence staining of FOXO3 in trained BMDMs for 12 h with or without alisertib. Scale bars: 10μM (left). The nuclear localization of FOXO3 was compared by calculated the ratio of mean nuclear intensity to cytoplasmic intensity and the representative data (right) showed the mean intensity of counted macrophages. (G) Western blot analysis of activation of AKT-mTOR-S6 pathway in β-glucan trained BMDM. BMDMs were transfected with smalling interferon RNA targeting AurA for 48 h, followed by β-glucan training for 6 h (left); BMDM was trained with β-glucan in the absence or presence of alisertib for 6 h (right). (H) Supernatant levels of IL-6 and TNF-α in BMDMs. The cells were treated with β-glucan training and aurora A inhibition in combination with mTOR agonist, MHY1485 (2 μM), and restimulated with MC38 culture supernatant for 24 h. Data are representative of three independent experiments and presented as the mean ± SEM. P values were derived from one-way ANOVA test with a Turkey’s multiple-comparison test (D, H) or with Dunnett’s multiple-comparison test (F). Related to Figure 5—source data 1-3.

Alisertib abrogates the anti-tumor effect induced by trained immunity.

(A) Experimental scheme of mouse experiment. 6∼8 weeks old mice was injected with β-glucan and administrated with alisertib, followed by 1×106 MC38 cells inoculation (n=5 per group). (B) Tumor growth curve of MC38 in mice as shown in A. (C) Flow cytometric analysis of myeloid cells (CD45+CD11b+) and macrophages (CD45+CD11b+F4/80+) cells in MC38 subcutaneous tumors in A. Gating strategy was shown in Fig S5C. (D) Co immunofluorescence staining of DAPI, F4/80 and GNMT tumor section; Scale bars: 20 μM. (E) FACs for intracellular phospho-S6 in gated macrophage. (F) Tumor tissue was lysed and the supernatant were collected for detection of cytokines production in tumor microenvironment. Data are represented as the mean ± SEM. P values were derived from one-way ANOVA test with Dunnett’s multiple-comparison test. Related to Figure 6—figure supplement 1, Figure 6— source data 1.

Targeting aurora A inhibits β-glucan-induced trained immunity.

(A) Schematic of the assay protocol for the molecule screening in BMDMs. (B) Fold change of IL-6 production in drug screening inhibited by AurA inhibitors compared to the β-glucan only group. (C) BMDMs were trained with β-glucan (50 μg/mL) in the presence of Tozasertib (1 μM), alisertib (1 μM) or MLN8054 (1 μM) for 24 h, followed by LPS (20 ng/mL) stimulation for 6 h after a rest for 3 days. (D) Supernatant levels of IL-6 in trained J774A.1 cells and THP-1 cells. J774A.1 cells were transfected with small interfering RNAs to knock down AurA followed by β-glucan (50 μg/mL) training. After rest for 3 days, trained J774A.1 cells was counted and seed into cell culture plate with LPS (20 ng/mL) rechallenge for 6 h. THP-1 cells were trained with β-glucan (50 μg/mL) for 24 h, and were centrifuged and washed once to remove medium. The THP-1 cells were then cultured with fresh medium containing 10 ng/mL PMA for 48 h and rest 1 day followed with LPS rechallenge (20 ng/mL). Data in C and D are representative of three independent experiments and presented as the mean ± SEM. P values were derived from one-way ANOVA test with a Dunnett’s multiple-comparison test. Related to Figure 1.

Aurora kinase A inhibition suppresses the expression of transcription factor involved in inflammation activation.

(A) In vivo training model in C57BL/6J mice with intraperitoneal injection of β-glucan (1 mg per mice) and daily administration of alisertib (30 mg/kg/d) for 7 days (mice n=2 per group). (B) Heatmap from RNA-seq analysis showing the differentially expressed transcription factors (DETFs) from mice treated as described in Figure S2A. (C) GO enrichment analysis of differentially expressed transcription factors (DETFs) between alisertib treated and untreated trained BMDMs. (D) Multiplex immunoassay measuring 18 cytokines/chemokines in supernatant from trained BMDMs as described in Figure S2A, which were rechallenged with LPS (20 ng/mL) for 6 h. Related to Figure 2.

Alisertib inhibits glucose incorporation into glycolysis and TCA cycle.

(A) Mass labelling of trained BMDMs in the absence or presence of alisertib for 24 h and administered U-13C-glucose simultaneously. Training with β-glucan increased incorporation of 13C-glucose into glycolysis and TCA cycle intermediates; this was reversed by alisertib. (B) peak area of tyrosine and comparing of the sum of peak areas for unlabeled and labeled tyrosine between different treatment groups. (C) Diagram illustrating the cross link between glycolysis, TCA cycle, glutathione and SAM. Data are representative of three independent experiments and presented as the mean ± SEM. P values were derived from one-way ANOVA (A) or two-way ANOVA (B) test with a Dunnett’s multiple-comparison test. Related to Figure 3.

Inhibition of Aurora kinase A impairs histone trimethylation at H3K4 and H3K36.

(A) quantification of H3K4m1, H3K9me3, H3K36me3, H3K4me3 and H3K27me3 protein level was determined by Image Lab software; data are represented as mean values ± SEM, P values were derived from one-way ANOVA with Turkey’s multiple-comparison test of n = 3 independent biological experiments. Related to Figure 4.

Alisertib abrogates the anti-tumor effect induced by trained immunity.

(A) Tumor growth curve for individual mice (n=5 per group). (B) Tumor images and tumor weight; data are represented as mean values ± SEM, P values were derived from one-way ANOVA with Dunnett’s multiple-comparison test. (C) The gating strategy used for analyzing tumor-infiltrating macrophages. Related to Figure 6.