Fig. 7

Irg1 Is Required for Enhanced mROS Production within Macrophage-Lineage Cells in Response to Fatty Acid Supplementation

Inhibition of β-oxidation reduces LPS-stimulated mROS production and bactericidal activity of J774.2 murine macrophage-like cells.

(A and B) Live confocal imaging of mROS production by macrophage-lineage cells within the midbrain/hindbrain region of control MO-injected Tg(mpeg1:EGFP) larvae, at 3 hpi, following Salmonella (Sal.)/MitoSOX injection alone or supplemented with an equimolar fatty acid mixture, respectively. MitoSOX fluorescence intensity is displayed as a colormap, with warmer colors representing higher signal intensities.

(C) Quantification of mROS production by macrophage-lineage cells (measured as total fluorescence intensities of MitoSOX within individual mpeg1⁺ cells), as marked in (A) and (B), within control MO-injected and Irg1-depleted Tg(mpeg1:EGFP) larvae at 3 hpi, following the indicated treatments (mean ± SD). Five to ten larvae were sampled for each treatment.

(D) Quantification of MitoSOX fluorescence, as detected by flow cytometry (measured as mean fluorescence intensity), within J774.2 murine macrophage-like cells treated with LPS alone (500 ng/ml) or supplemented with 50 µM etomoxir, compared to untreated, n = 4 biological replicates (mean ± SD).

(E) Gentamicin protection assay showing infected DAPI-stained J774.2 cells (either untreated (control) or treated with 50 µM etomoxir) at 2, 8, and 24 hr postinfection (hpi) with Sal-GFP.

(F) Quantification of recovered Sal-GFP following cell lysis and plating of samples, as shown in (E), n = 3 biological replicates (mean ± SD).

(G) Model for Irg1/β-oxidation-dependent bactericidal mROS production. Scale bars, 5 µm in (A) and (E). Abbreviations: n.s., not significant; p < 0.05; p < 0.001; p < 0.0001. See also Figure S7.