Fig. 8

Macrophages are activated during transfusion-associated necrotizing enterocolitis (NEC). a Scatter bar diagrams show nuclear factor-κB (NF-κB) activity in reporter cells treated with nanoparticles (NPs) containing small interfering RNA (siRNA) against NF-κB p65 for 24 h, followed by 7-day-stored red blood cell (RBCs). Controls included no NPs or NPs containing scrambled siRNA; N = 8 donors; Kruskal–Wallis H test with Dunn’s post test, ^†P < 0.001. Fluorescence photomicrographs (magnification ×2400) show “activated” appearance and nuclear translocation of phospho-NF-κB p65 (green) in a macrophage treated with control NPs. Cells treated with anti-p65 NPs did not show phospho-NF-κB p65. Arrows indicate NPs. Scale bar = 5 µm. b Kaplan–Meier curves show survival without intestinal injury in anemic-transfused pups treated with control or anti-p65 NPs. NPs with anti-p65 siRNA were protective; Mantel–Cox log-rank test, ^†P < 0.001. c Photographs show intestinal injury in an anemic-transfused pup treated with control NPs (arrows), but no injury in a pup treated with NPs containing anti-p65 siRNA. Scatter plots below summarize the severity of bowel injury in the two groups. N = 6 anemic-transfused mice treated with control NPs, 11 anemic-transfused treated with anti-p65 NPs; Mann–Whitney U test, ^†P < 0.001. d Fluorescence photomicrographs (magnification ×400) of proximal colon from anemic-transfused mice treated with either control or anti-p65 NPs show immunoreactivity for Ly6C (magenta), fluorescence tag on NPs (red), and phospho-NF-κB p65 (green). Mice treated with control NPs show tissue damage and nuclear translocation of phospho-p65, whereas those treated with anti-p65 NPs show preserved histoarchitecture and no immunoreactivity for phospho-p65 in Ly6C⁺ macrophages. Cytoplasmic staining for phospho-p65 was seen in two cells (open arrows) that did not express Ly6C. Scale bar = 50 µm. High-magnification photomicrographs on right (×2000; phospho-p65 staining in area in boxes) highlight nuclear localization (arrow) of phospho-p65 in the control NP group; N = 5 mice per group. All scatter plots summarize the data as means ± standard error of the mean