Transfusion of stored blood impairs host defenses against Gram-negative pathogens in mice

Abstract

Background: Although human red blood cell (RBC) units may be refrigerator stored for up to 42 days, transfusion of older RBCs acutely delivers a large bolus of iron to mononuclear phagocytes. Similarly, iron dextran circulates in plasma for hours to days and is progressively cleared by mononuclear phagocytes, which return iron to plasma. Finally, malaria infection continuously delivers iron to macrophages by intra- and extravascular hemolysis. Studies suggest that iron administration increases infectious risk.

Study design and methods: To assess the effects of increased iron availability on susceptibility to infection, we infected mice with model Gram-negative intracellular or extracellular pathogens (Salmonella typhimurium or Escherichia coli, respectively), accompanied by RBC transfusion, iron dextran administration, or malarial coinfection.

Results: In our mouse models, transfusion of older RBCs exacerbates infection with both Gram-negative pathogens. Although iron dextran exacerbates E. coli infection to a similar extent as transfusion of corresponding amounts of iron, higher iron doses are required to produce comparable effects with S. typhimurium. Coinfection of mice with Plasmodium yoelii and S. typhimurium produces overwhelming Salmonella sepsis. Finally, treating mice with antibiotics abrogates the enhancing effect on E. coli infection of both older RBC transfusion and iron dextran administration.

Conclusions: Transfusion of older RBCs exacerbates Gram-negative infection to a similar extent as malaria coinfection or iron dextran administration. Appropriate antibiotic therapy abrogates the effect of older RBC transfusions on infection with E. coli. Iron delivery to macrophages may be an underappreciated mechanism mediating, at least some, adverse effects of RBC transfusions.

Fig. 1

Older, stored RBC transfusions exacerbate sepsis and decrease survival in S. typhimurium–infected mice. Transfusion recipients were male C57BL/6 mice. (A) Mice were infected intraperitoneally with 1000 CFUs of S. typhimurium strain LT2 and infused with saline (n = 35; black circles); fresh syngeneic RBCs (<24-hr storage; n = 39; blue squares); or older, stored RBCs (2 weeks of storage; n = 28; red triangles). Kaplan-Meier survival estimates of mice are shown. (B–D) Salmonella infected mice (n = 10 per group) were infused with saline, fresh RBCs, or old RBCs, as above, and euthanized 5 days later. Bacterial load was measured by plate dilution in (B) blood, (C) liver, and (D) spleen. Results are presented as mean ± SEM; no bacteria were detected in the blood of mice infused with saline or transfused fresh RBCs. (E) Representative images of hematoxylin and eosin–stained histologic sections of the liver and spleen, 5 days after transfusion of S. typhimurium –infected mice with fresh or old RBCs or mice transfused old RBCs without infection. The arrow denotes tissue necrosis; the arrowhead denotes a blood vessel. Original magnification was 400× (liver) and 40× (spleen). ***p < 0.001 compared to fresh RBC transfusions.

Fig. 2

S. typhimurium infection is exacerbated by coinfection with malaria or iron delivery to macrophages. (A) C57BL/6 mice were infected intraperitoneally with approximately 100,000 P. yoelii merozoites with or without infection with 1000 CFUs of S. typhimurium strain LT2. Kaplan-Meier survival estimates are shown. (B) Parasitemia, measured by microscopic evaluation of 100 Wright Giemsa–stained RBCs obtained from the tail vein, was determined by a blinded observer at each indicated time point; no significant differences were seen. (C) C57BL/6 mice were infected intraperitoneally with 1000 CFUs of S. typhimurium strain LT2 and infused IV with dextran (70,000 MW; 20 mg) or iron dextran (from 0.5–4 mg iron). Kaplan-Meier survival estimates are shown. **p < 0.01, ***p < 0.001 for the comparisons indicated.

Fig. 3

Iron delivery to macrophages or rapid clearance of transfused RBCs exacerbates E. coli infection. CD-1 mice were used in these experiments. (A) Mice were infused with saline (n = 26; black circles); fresh syngeneic RBCs (<24-hr storage; n = 31; blue squares); older, stored RBCs (2 weeks of storage; n = 36; red triangles); dextran (20 mg; 70,000 MW; n = 25); or iron dextran (0.5 mg of iron; n = 25). Mice were concurrently infected intraperitoneally with E. coli strain Xen 14 (approx. 1 × 10⁸ CFUs). Kaplan-Meier survival estimates are shown. (B) Mice were infused with saline (black circles); syngeneic fresh RBCs (blue squares); older, stored RBCs (red triangles); washed, older, stored RBCs (orange diamonds); supernatant derived from older, stored RBCs (light blue triangles); or ghosts prepared from older, stored RBCs (green stars). Mice were concurrently infected intraperitoneally with E. coli strain Xen 14 (approx. 1 × 10⁸ CFUs). Kaplan-Meier survival estimates are shown. *p < 0.05, ***p < 0.001, as indicated.

Fig. 4

The effect of old RBC transfusions on exacerbating infection is ameliorated by antibiotic treatment. Experiments were performed using CD-1 mice, treated with or without a fluoroquinolone antibiotic (i.e., enrofloxacin). (A) Mice were transfused with syngeneic fresh RBCs (<24-hr storage) or old RBCs (2 weeks of storage) and concurrently infected intraperitoneally with E. coli strain Xen 14 (approx. 1 × 10⁸ CFUs). Kaplan-Meier survival estimates are shown. (B) Mice were infused with dextran (20 mg; 70,000 MW) or iron dextran (0.5 mg iron) and concurrently infected intraperitoneally with E. coli strain Xen 14 (approx. 1 × 10⁸ CFUs). Kaplan-Meier survival estimates are shown. (C) Representative bioluminescence images of mice at defined time points after infection, as described above. Red X’s represent animal death. (D) Bioluminescence was quantified in a defined region over the abdomen of each mouse and the area under the curve (AUC) was calculated for the first 12 hours (i.e., before animal dropout due to death or euthanasia for severe morbidity). *p < 0.05, **p < 0.01, ***p < 0.001, as indicated.