Lipopolysaccharide alters iron homeostasis in both wild type and β-thalassemia mice even under parenteral iron loading

Background: Hepcidin controls iron homeostasis by inducing the degradation of the iron eﬄux protein, ferroportin (FPN1), and subsequently reducing serum iron levels. Hepcidin expression is inﬂuenced by multiple factors, including iron stores, ineﬀective erythropoiesis, and inﬂammation. However, the interactions between these factors under thalassemic condition remain unclear. This study aimed to determine the hypoferremic and transcriptional responses of iron homeostasis to acute inﬂammatory induction by lipopolysaccharide (LPS) in thalassemic ( Hbb th3 /+ ) mice with/without parenteral iron loading with iron dextran. Methods: Wild type and Hbb th3 /+ mice were intramuscularly injected with 5 mg of iron dextran once daily for 2 consecutive days. After a 2-week equilibration, acute inﬂammation was induced by a peritoneal injection of a single dose of 1 µg/g body weight of LPS. Control groups for both iron loading and acute inﬂammation received equal volume(s) of saline solution. Blood and tissue samples were collected at 6 hours after LPS (or saline) injection. Iron parameters and mRNA expression of hepcidin as well as genes involved in iron transport and metabolism in wild type and Hbb th3 /+ mice were analyzed and compared by Kruskal-Wallis test with pairwise Mann-Whitney U test. Results: We found the inductive eﬀects of LPS on liver IL-6 mRNA expression to be more pronounced under parenteral iron loading. Upon LPS administration, splenic erythroferrone (ERFE) mRNA levels were reduced only in iron-treated mice, whereas, liver bone morphogenetic protein 6 (BMP6) mRNA levels were decreased under both control and parenteral iron loading conditions. Despite the altered expression of the aforementioned hepcidin regulators, the stimulatory eﬀect of LPS on hepcidin mRNA expression was blunt in iron-treated Hbb th3 /+ mice. Contrary to the blunted hepcidin response, LPS treatment suppressed FPN1 mRNA expression in the liver, spleen, and duodenum, as well as reduced serum iron levels of Hbb th3 /+ mice with parenteral iron loading. Conclusion: Our study suggests that a hypoferremic response to LPS-induced acute inﬂammation is maintained in thalassemic mice with parenteral iron loading in a hepcidin-independent manner. Abstract 31 Background: Hepcidin controls iron homeostasis by inducing the degradation of the iron efflux 32 protein, ferroportin (FPN1), and subsequently reducing serum iron levels. Hepcidin expression 33 is influenced by multiple factors, including iron stores, ineffective erythropoiesis, and 34 inflammation. However, the interactions between these factors under thalassemic condition 35 remain unclear. This study aimed to determine the hypoferremic and transcriptional responses of 36 iron homeostasis to acute inflammatory induction by lipopolysaccharide (LPS) in thalassemic 37 ( Hbb th3 mice with/without parenteral iron loading with iron dextran. 38 Methods: Wild type and Hbb th3 /+ mice were intramuscularly injected with 5 mg of iron dextran 39 once daily for 2 consecutive days. After a 2-week equilibration, acute inflammation was induced 40 by a peritoneal injection of a single dose of 1 µg/g body weight of LPS. Control groups for both 41 iron loading and acute inflammation received equal volume(s) of saline solution. Blood and 42 tissue samples were collected at 6 hours after LPS (or saline) injection. Iron parameters and 43 mRNA expression of hepcidin as well as genes involved in iron transport and metabolism in wild 44 type and Hbb th3 /+ mice were analyzed and compared by Kruskal-Wallis test with pairwise Mann-45 Whitney U test. 46 Results: We found the inductive effects of LPS on liver IL-6 mRNA expression to be more 47 pronounced under parenteral iron loading. Upon LPS administration, splenic erythroferrone (ERFE) mRNA levels were reduced only in iron-treated mice, whereas, liver bone morphogenetic under both control and parenteral iron loading conditions. altered expression of the aforementioned response, LPS treatment suppressed 53 FPN1 mRNA expression in the liver, spleen, and duodenum, as well as reduced serum iron levels 54 of Hbb th3 /+ mice with parenteral iron loading. 55 Conclusion: Our study suggests that a hypoferremic response to LPS-induced acute 56 inflammation is maintained in thalassemic mice with parenteral iron loading in a hepcidin-57 independent manner. model expression hepcidin iron normalized to β-actin ( Actb ) expression. SEM the fold compared WT (WT (n=5 Kruskal-Wallis test pairwise Mann-Whitney test. acquired values subsequently Bonferroni