Fig. 6

Red blood cell (RBC) transfusions cause intestinal injury by activating Toll-like receptor-4 (TLR4). a Scatter bar diagrams show fold change in Tlr4 and myeloid differentiation primary response gene 88 (Myd88) mRNA expression in the intestinal tissue from control and anemic-transfused mice. Kruskal–Wallis H test with Dunn’s post test, *P < 0.05, **P < 0.01 vs. naive control. N = 8 mice per group. b RBCs used for transfusion activate TLR4. Scatter bar diagram shows the activation of a reporter gene (secreted embryonic alkaline phosphatase) upon stimulation with 7-day-stored RBCs in TLR4-reporter cells. Mann–Whitney U test, ^†P < 0.001 vs. naive control. N = 8 different donor mice. c RBCs used for transfusion fail to stimulate TNF production in RAW264.7 macrophages with Tlr4 and Myd88 knockdown. Ticam1 knockdown did not block TNF production. Kruskal–Wallis H test with Dunn’s post test, **P < 0.01 vs. control. N = 8 independent biological replicates per group. d Scatter plots show severity of transfusion-associated bowel injury in anemic-transfused Tlr4 (−/−) pups vs. wild-type (WT) littermates, in the ileum (left) and colon (right). Representative photographs above show ileal and colonic injury in WT, but not in the Tlr4 (−/−) intestine. N = 8 Tlr4 (−/−), 9 WT mice; Mann–Whitney U test, **P < 0.01, ^†P < 0.001 vs. WT; e Kaplan–Meier curves summarize survival without intestinal injury in anemic-transfused Tlr4 (−/−) pups vs. WT littermates; Mantel–Cox log-rank test, ^†P < 0.001. f Scatter plots summarize Il1b (top) and Tnf (bottom) expression in ileum from anemic-transfused Tlr4 (−/−) pups vs. WT littermates, expressed as fold change over naive control of the same genotype. N = 3 Tlr4 (−/−), 4 WT mice; Mann–Whitney U test, *P < 0.05, **P < 0.01. g Representative flow cytometry scatter plots of the CD11b-positive myeloid cell fraction in the anemic and anemic-transfused Tlr4 (−/−) intestine show a F4/80 (mid) CD115 (+) Ly6C (+) cell population. Hematoxylin and eosin (H&E)-stained photomicrographs of the colon from anemic and anemic-transfused Tlr4 (−/−) mice show increased cellularity (arrows) in the lamina propria. Scale bars = 50 µm. N = 8 Tlr4 (−/−), 9 WT mice. All scatter plots summarize the data as means ± standard error of the mean