Exploring the effect of iron ligand interactions on iron uptake pathways and inflammatory response in human cell lines
Doctoral thesis, 2025
Iron supplementation is essential for treatment of iron deficiency anemia, the most common nutritional disorder worldwide (WHO, 2021). While oral iron supplementation remains the first-line therapy, its use is not without risk, particularly for individuals with pre-existing gastrointestinal conditions such as inflammatory bowel disease (IBD) or Crohn’s disease, where pro-inflammatory responses to certain iron formulations could exacerbate the condition. Understanding how the iron-ligand interactions of oral iron compounds influence their inflammatory potential is critical for optimizing treatment safety and efficacy.
Investigating six iron chelates and six iron salts demonstrated that all iron chelates significantly induced the MAPK signaling pathway, as evidenced by elevated amphiregulin levels (Paper I). The two most chemically stable polydentate chelates, ferric EDTA and ferric pyrophosphate, upregulated amphiregulin and IGFr1 at physiologically relevant concentrations ([Fe]=0.05 mM). This effect was not observed with any of the tested iron salts. Ferric pyrophosphate also induced the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) and stimulated production of interleukin-6, indicating the most pronounced pro-inflammatory profile among the tested compounds.
To further investigate these inflammatory responses, we examined COX-2 and lipooxygenase-5 (LOX-5) levels in intestinal cell lines derived from both male and female donors (Paper III). The results demonstrated that solubility and the particle size, formed in cellular medium (pH 7.4) supplemented with ferric pyrophosphate or ferrous fumarate was associated with elevated COX-2 levels in most of the tested cell lines. The fully soluble ferric EDTA did not elicit such response. Importantly, these effects occurred independently of ferritin accumulation, suggesting that inflammatory signaling was driven by the formed particles, rather than by total cellular iron load.
In a mechanistic follow-up study (Paper II), ferrous fumarate was found to utilize both the DMT1 transporter and clathrin-mediated endocytosis for cellular uptake, challenging the common view that the intestinal uptake of iron from iron salts is tightly regulated by means of being transported by the DMT1-mediated pathway. Cellular uptake of iron from ferrous sulfate and ferric EDTA was confirmed to be independent of clathrin-mediated endocytosis. In a gut-liver axis model (Caco-2/HepG2), ferric pyrophosphate treatment (0.2 mM) induced key MAPK/ERK pathway activators, including the IFN-γ receptor, EGF, and CDKN1A, which was further validated in Caco-2 monocultures exposed to commercially available ferric pyrophosphate (0.4 mM), where IL-6 production was observed. This response was not observed in cells treated with a nanoparticulate iron, Iron hydroxide adipate tartrate (IHAT).
In conclusion, this thesis demonstrates that the iron-ligand interactions are critical determinants of both the intestinal uptake route and downstream inflammatory effects in intestinal cell models. These insights highlight the need to carefully consider the choice of iron ligand, when designing an iron supplements in order to minimize adverse effects while ensuring therapeutic efficacy.
Investigating six iron chelates and six iron salts demonstrated that all iron chelates significantly induced the MAPK signaling pathway, as evidenced by elevated amphiregulin levels (Paper I). The two most chemically stable polydentate chelates, ferric EDTA and ferric pyrophosphate, upregulated amphiregulin and IGFr1 at physiologically relevant concentrations ([Fe]=0.05 mM). This effect was not observed with any of the tested iron salts. Ferric pyrophosphate also induced the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) and stimulated production of interleukin-6, indicating the most pronounced pro-inflammatory profile among the tested compounds.
To further investigate these inflammatory responses, we examined COX-2 and lipooxygenase-5 (LOX-5) levels in intestinal cell lines derived from both male and female donors (Paper III). The results demonstrated that solubility and the particle size, formed in cellular medium (pH 7.4) supplemented with ferric pyrophosphate or ferrous fumarate was associated with elevated COX-2 levels in most of the tested cell lines. The fully soluble ferric EDTA did not elicit such response. Importantly, these effects occurred independently of ferritin accumulation, suggesting that inflammatory signaling was driven by the formed particles, rather than by total cellular iron load.
In a mechanistic follow-up study (Paper II), ferrous fumarate was found to utilize both the DMT1 transporter and clathrin-mediated endocytosis for cellular uptake, challenging the common view that the intestinal uptake of iron from iron salts is tightly regulated by means of being transported by the DMT1-mediated pathway. Cellular uptake of iron from ferrous sulfate and ferric EDTA was confirmed to be independent of clathrin-mediated endocytosis. In a gut-liver axis model (Caco-2/HepG2), ferric pyrophosphate treatment (0.2 mM) induced key MAPK/ERK pathway activators, including the IFN-γ receptor, EGF, and CDKN1A, which was further validated in Caco-2 monocultures exposed to commercially available ferric pyrophosphate (0.4 mM), where IL-6 production was observed. This response was not observed in cells treated with a nanoparticulate iron, Iron hydroxide adipate tartrate (IHAT).
In conclusion, this thesis demonstrates that the iron-ligand interactions are critical determinants of both the intestinal uptake route and downstream inflammatory effects in intestinal cell models. These insights highlight the need to carefully consider the choice of iron ligand, when designing an iron supplements in order to minimize adverse effects while ensuring therapeutic efficacy.
precipitation
Hutu-80
inflammation
iron ligand
non-heme iron
intestinal epithelial cells
iron chelates
dietary iron
solubility
iron uptake
iron salts
Caco-2
10:an
Opponent: Alida Melse-Boonstra, Wageningen University, Netherlands
Author
Agata Tarczykowska
Chalmers, Life Sciences, Food and Nutrition Science
Uptake of iron from ferrous fumarate can be mediated by clathrin-dependent endocytosis in Hutu-80 cells
Frontiers in Molecular Biosciences,;Vol. 12(2025)
Journal article
Differential Effects of Iron Chelates vs. Iron Salts on Induction of Pro-Oncogenic Amphiregulin and Pro-Inflammatory COX-2 in Human Intestinal Adenocarcinoma Cell Lines
International Journal of Molecular Sciences,;Vol. 24(2023)
Journal article
Tarczykowska A., Mohammadi A.S., Scheers N. COX-2 induction in response to the iron compounds ferric pyrophosphate and ferrous fumarate in human intestinal cell lines (manuscript).
Tarczykowska A., Scheers N. Comparative Effects of Ferric Pyrophosphate and IHAT on MAPK/ERK Pathway mediators in single-cultured Caco-2 cells and in the Caco-2/HepG2 co-culture model (manuscript).
Iron deficiency anemia remains the most prevalent nutritional disorder affecting 25% of the global population, especially women, children and individuals with certain health conditions (IBS, Crohn’s disease). Oral iron supplementation continues to be the primary treatment, however it is still not fully known how different iron compounds interact with the bowels and ultimately with the whole body and what physiological responses they may trigger.
This thesis offers new insights into how iron-ligand interactions influence inflammatory response in intestinal cells. The findings show that iron chelates, particularly ferric pyrophosphate, a commercially available iron supplement, can activate signaling pathways linked to inflammation and cancer growth in intestinal cell models. The observed effects occur independently of total intracellular iron load. Moreover, solubility and particle formation by the iron compounds also affected cellular response: poorly soluble ones, ferrous fumarate and ferric pyrophosphate, lead to formation of relatively large particles in the cell culture medium, which in turn was associated with induction of the pro-inflammatory mediator cyclooxygenase 2 in human intestinal cells.
This work challenges the common view that intestinal uptake of (non-heme bound) iron is tightly regulated and mainly absorbed by the iron transporter DMT1. This thesis shows that the common iron salt, ferrous fumarate, can be absorbed in its insoluble form, by means of endocytosis, previously only considered as an uptake route for designed supplemental forms of nanoparticulate iron. The new view of a less regulated iron uptake implies that we need to carefully consider iron-ligand effects when designing new supplemental iron forms and consider smaller dosing, to avoid unwanted pro-inflammatory and pro-oncogenic effects.
This thesis offers new insights into how iron-ligand interactions influence inflammatory response in intestinal cells. The findings show that iron chelates, particularly ferric pyrophosphate, a commercially available iron supplement, can activate signaling pathways linked to inflammation and cancer growth in intestinal cell models. The observed effects occur independently of total intracellular iron load. Moreover, solubility and particle formation by the iron compounds also affected cellular response: poorly soluble ones, ferrous fumarate and ferric pyrophosphate, lead to formation of relatively large particles in the cell culture medium, which in turn was associated with induction of the pro-inflammatory mediator cyclooxygenase 2 in human intestinal cells.
This work challenges the common view that intestinal uptake of (non-heme bound) iron is tightly regulated and mainly absorbed by the iron transporter DMT1. This thesis shows that the common iron salt, ferrous fumarate, can be absorbed in its insoluble form, by means of endocytosis, previously only considered as an uptake route for designed supplemental forms of nanoparticulate iron. The new view of a less regulated iron uptake implies that we need to carefully consider iron-ligand effects when designing new supplemental iron forms and consider smaller dosing, to avoid unwanted pro-inflammatory and pro-oncogenic effects.
Subject Categories (SSIF 2025)
Molecular Biology
Pharmacology and Toxicology
Areas of Advance
Life Science Engineering (2010-2018)
ISBN
978-91-8103-264-2
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5722
Publisher
Chalmers
10:an
Opponent: Alida Melse-Boonstra, Wageningen University, Netherlands