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. 2023 Jun 12;142(6):589–606. doi: 10.1182/blood.2022019145

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Figure 4. — RvD4 stops neutrophil emergency deployment and accelerates the resolution of infection. Mice inoculated with E coli (10⁵ CFU, IP) were also administered (IV) with RvD4 (100 ng per mouse) or vehicle alone (0.01% v/v ethanol in saline). Exudates, whole blood, and BM cells were collected at 0, 12, and 72 hours. (A) Schematic of the experimental design and sample collection. (B) CyTOF: UMAP of the infectious exudate, whole blood, and the BM, labeled with 17 immune populations. (C) Number of neutrophils in exudate, whole blood, and the BM. (D) Number of LSKs and GMPs in whole blood and the BM. (E) UMAP of neutrophil populations and progenitors in the BM, whole blood, and exudate. (F) Neutrophil lineage trajectory: diffusion map. LSKs, GMPs, PN (preneutrophils), IN (immature neutrophils), MN (mature neutrophils); and CN, circulating neutrophils. (G) RvD4 and E coli logFC change diffusion map calculated using edgeR with diffcyt. Only statistically significant populations are colored (P < .05) and adjusted using a Benjamini-Hochberg correction. Results in panels C-D are mean ± SEM; n = 3 samples biologically independent per time point. RvD4 vs E coli + vehicle; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001 using 1-way ANOVA with Tukey multiple comparison test. (H) Exudate: E coli bacterial titers (300 μL lavage) at 12 hours. Results are expressed as mean ± SEM; n = 8 or 9 samples per condition. RvD4 vs E coli + vehicle, ∗∗P < .01 using 2-tailed t test. (I-J) Exudate: in vivo phagocytosis. Intracellular E coli levels were determined in neutrophils (CD45⁺F4/80⁻Ly6C⁻Ly6G⁺), monocytes (CD45⁺F4/80⁻Ly6G⁻Ly6C⁺), and macrophages (CD45⁺Ly6G⁻Ly6C⁻F4/80⁺). MFI, mean fluorescence intensity. (I) Representative histograms. (J) Quantification of MFI of intracellular E coli levels. Results are expressed as mean ± SEM; n = 4 samples; RvD4 vs E coli + vehicle, ∗P < .05 using 2-way ANOVA with Bonferroni multiple comparisons test. (K-L) Flow cytometry: exudate neutrophil apoptosis (CD45⁺Ly6G⁺annexinV⁺) at 12 hours. (K) Representative dot plots and (L) quantification of neutrophil apoptosis. Results are expressed as mean ± SEM; n = 4 or 5 samples. RvD4 vs E coli + vehicle, ∗∗∗∗P < .0001 using 2-way ANOVA with Bonferroni multiple comparisons test. (M) Flow cytometry: exudate in vivo macrophage efferocytosis (CD45⁺CD11b⁺F4/80⁺Ly6G⁺) at 12 hours. Results are expressed as mean ± SEM; n = 4 or 5 samples per condition. RvD4 vs E coli + vehicle, ∗P < .05 using 2-tailed t test.

Figure 4. — RvD4 stops neutrophil emergency deployment and accelerates the resolution of infection. Mice inoculated with E coli (10⁵ CFU, IP) were also administered (IV) with RvD4 (100 ng per mouse) or vehicle alone (0.01% v/v ethanol in saline). Exudates, whole blood, and BM cells were collected at 0, 12, and 72 hours. (A) Schematic of the experimental design and sample collection. (B) CyTOF: UMAP of the infectious exudate, whole blood, and the BM, labeled with 17 immune populations. (C) Number of neutrophils in exudate, whole blood, and the BM. (D) Number of LSKs and GMPs in whole blood and the BM. (E) UMAP of neutrophil populations and progenitors in the BM, whole blood, and exudate. (F) Neutrophil lineage trajectory: diffusion map. LSKs, GMPs, PN (preneutrophils), IN (immature neutrophils), MN (mature neutrophils); and CN, circulating neutrophils. (G) RvD4 and E coli logFC change diffusion map calculated using edgeR with diffcyt. Only statistically significant populations are colored (P < .05) and adjusted using a Benjamini-Hochberg correction. Results in panels C-D are mean ± SEM; n = 3 samples biologically independent per time point. RvD4 vs E coli + vehicle; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001 using 1-way ANOVA with Tukey multiple comparison test. (H) Exudate: E coli bacterial titers (300 μL lavage) at 12 hours. Results are expressed as mean ± SEM; n = 8 or 9 samples per condition. RvD4 vs E coli + vehicle, ∗∗P < .01 using 2-tailed t test. (I-J) Exudate: in vivo phagocytosis. Intracellular E coli levels were determined in neutrophils (CD45⁺F4/80⁻Ly6C⁻Ly6G⁺), monocytes (CD45⁺F4/80⁻Ly6G⁻Ly6C⁺), and macrophages (CD45⁺Ly6G⁻Ly6C⁻F4/80⁺). MFI, mean fluorescence intensity. (I) Representative histograms. (J) Quantification of MFI of intracellular E coli levels. Results are expressed as mean ± SEM; n = 4 samples; RvD4 vs E coli + vehicle, ∗P < .05 using 2-way ANOVA with Bonferroni multiple comparisons test. (K-L) Flow cytometry: exudate neutrophil apoptosis (CD45⁺Ly6G⁺annexinV⁺) at 12 hours. (K) Representative dot plots and (L) quantification of neutrophil apoptosis. Results are expressed as mean ± SEM; n = 4 or 5 samples. RvD4 vs E coli + vehicle, ∗∗∗∗P < .0001 using 2-way ANOVA with Bonferroni multiple comparisons test. (M) Flow cytometry: exudate in vivo macrophage efferocytosis (CD45⁺CD11b⁺F4/80⁺Ly6G⁺) at 12 hours. Results are expressed as mean ± SEM; n = 4 or 5 samples per condition. RvD4 vs E coli + vehicle, ∗P < .05 using 2-tailed t test.