Fig 2: Functional alteration of neutrophils in serum iron deficiency.(A) Phenotyping ex vivo of isolated BM neutrophils (obtained from mice treated as in Fig. 2A). Dihydrorhodamine 123 (DHR123) fluorescence as a reporter for cytoplasmic ROS in PMA-stimulated cells. Phagocytosis of GFP-labeled E. coli. FMO, fluorescence minus one. Two-way ANOVA. Mean, quartiles, and range. Bacterial killing of S. aureus, resolved as colony-forming units of S. aureus recovered after coculture with neutrophils. Unpaired t test. Means ± SD. (B) Supernatant CCL2 and TNF levels produced by zymosan-stimulated neutrophils measured by enzyme-linked immunosorbent assay (ELISA). Unpaired t test. Mean, quartiles, and range. (C) NETosis was evaluated in isolated BM neutrophils after stimulation ex vivo with PMA and ionomycin ± DPI. A minimum of 250 cells from two replicates per sample counted. Two-way ANOVA. Mean, quartiles, and range. Representative microscopy images taken at ×20 magnification. (D) NETosis was evaluated in isolated BM neutrophils after stimulation ex vivo with PMA ± apocynin (APO). A minimum of 250 cells from two replicates per sample counted. Two-way ANOVA. Mean, quartiles, and range. Representative microscopy images taken at ×20 magnification. (E) SCENITH (single-cell metabolism by profiling translation inhibition) analysis of Ly6G+ BM neutrophils from experiment described in Fig. 1A, analyzing proportional shifts in O-propargyl-puromycin (OPP) incorporation after ex vivo treatment with metabolic inhibitors as detailed in Materials and Methods. Two-way ANOVA with matching for sample. Reported P value is the effect of mHep treatment. Mean. FAO, fatty acid oxidation; AAO,amino acid oxidation. (F) MitoSOX fluorescence as a reporter for mitoROS in ionomycin stimulated isolated BM neutrophils. Two-way ANOVA. Mean, quartiles, and range. (G) NETosis was evaluated in isolated BM neutrophils after stimulation ex vivo with ionomycin (iono) ± mitoTEMPO. A minimum of 250 cells from two replicates per sample counted. Two-way ANOVA. Mean, quartiles, and range. Representative microscopy images taken at ×20 magnification.

Fig 3: Anti-IL-6 antibodies dampened the protumour activities of ‘educated’ macrophages towards colon cells. (A)The level of IL-6, IL-12 and IL-23 in the colonic tissues were measured in mice from each group at 18 weeks after AOM/DSS treatment using ELISA (n = 5). (B) Western blot analysis of IL-6 levels in macrophages cocultured with miR-126-overexpressing or miR-126-silenced colon cells (n = 3). (C) Model of the coculture system of TAMs and colon cells. TAMs were first cocultured with miR-126-overexpressing or miR-126-silenced colon cells for 2 days. The cocultured TAMs were then removed from the previous system and placed in a new coculture system with untreated colon cells, and IL-6 neutralising antibodies (Abs, 100 ng·mL-1) were subsequently added. (D) CCK-8 assays were performed to determine cell growth (n = 5). (E) Transwell assays were performed to measure the cellular recruitment capacity, and the numbers of migrated cells were counted (right panel). Macrophages were cocultured with miR-126-overexpressing or miR-126-silenced colon cells, and IL-6 neutralising antibodies were added (magnification: 200×; scale bar = 50 µm, n = 8). (F) Representative images of immunofluorescence staining for E-cadherin (red), Vimentin (green) and DAPI (blue) (magnification: 400×; scale bar = 20 µm, n = 7, left panel). The mean immunofluorescence staining density of E-cadherin (upper right panel) and Vimentin (lower right panel) was measured (magnification: 400×; scale bar = 20 µm, n = 7). Individual data are presented as the mean ± SD. Statistical analyses were performed using unpaired t tests (*P < 0.05; **P < 0.01; and ***P < 0.0001).

Fig 4: Exogenous CoA induces a pro-inflammatory response in BMDMs(a) Volcano plot from bulk RNA sequencing data from BMDMs treated IL-4 (20 ng/mL), IL-4 + 1 mM CoA, or vehicle control for 48 hr. comparing differential gene expression between IL-4 vs. vehicle controls (left) and IL-4 + CoA vs. IL-4 (right). Genes associated with classical activation are depicted in red, genes associated with alternative activation are shown in blue. (b) Gene Set Enrichment Analysis of genes upregulated in BMDMs treated with IL-4+CoA vs. IL-4 alone. (c) qPCR analysis of Il1b, Tnf, Irg1, and Nos2 in BMDMs stimulated with CoA (1mM) or vehicle control for 4 hr. (n=4 independent biological replicates). (d) qPCR analysis of the interferon-stimulated gene Mx1 in BMDMs stimulated with 1 mM CoA or vehicle control for 24 hr. (n=4 independent biological replicates). (e) Itaconate abundance after treatment with 1 mM CoA or vehicle control for 48 hr. (n=6 independent biological replicates). (f) qPCR analysis of Il1b and Tnf in the peritoneal exudate cells of mice treated with (40 mg/kg) CoA 6 hr. prior to collection (n≥5 mice for each group). (g) Quantification of cytokines in the peritoneal lavage fluid (PLF) of mice treated as in (g) using Multiplexed ELISA (n=3 mice were used for each group). All data are presented as mean ± SEM. *p < 0.05; **p < 0.01.

Fig 5: CoA is a TLR4 agonist(a) qPCR analysis of Il1b, Tnf, Irg1, l12b, and Nos2 in WT and Myd88−/− BMDMs stimulated with 1 mM CoA or vehicle control for 4 hr. (n≥3 independent biological replicates). (b) Concentration-response curve of linked alkaline phosphatase activity in hTLR4 reporter cells with varying concentrations of LPS (black dots). The red dot and dashed lines represent the OD630 observed in response to 1 mM CoA treatment (n=4 independent biological replicates). (c) Aggregated response of 1 mM CoA compared to vehicle control in hTLR2, hTLR4, and hTLR7 relative to maximum TLR activation. (n≥3 independent biological replicates). (d) qPCR analysis of Il1b, Tnf, l12b, and Nos2 in WT and Tlr4−/− BMDMs following treatment with 1 mM CoA for 4 hr. (n=3 independent biological replicates). (e) Abundance of itaconate in WT, Myd88−/−, and Tlr4−/− BMDMs in response to 1 mM CoA treatment for 48 hr. or vehicle control (n≥5 independent biological replicates). All data are presented as mean ± SEM. *p < 0.05; ***p < 0.001.