Alveolar macrophages (AM) are critical to defense against respiratory pathogens. This study evaluated cellular iron imbalance to immunometabolism in endotoxin-polarized porcine AMs (PAMs). PAMs collected from five 6-week-old pigs were treated with a basal media, iron chelator, or ferric ammonium citrate to maintain iron replete or induce iron deficiency or overload, respectively. After 24 h treatment, PAMs were challenged with saline or lipopolysaccharide (LPS) for 6 h. Cells were analyzed for gene, protein, and untargeted metabolome. Cytokines were determined in culture media. Data were assessed using two-way ANOVA. Treatments successfully induced iron deficiency and overload. The mRNA of DMT1 and ZIP14 was increased up to 300-fold by LPS, but unaffected by iron. Surprisingly, both iron deprivation and overload attenuated LPS-induced inflammation, showing less TNFα production and lower mRNA of pro- and anti-inflammatory cytokines than iron-replete PAMs. Forty-eight metabolites were altered by either or both main effects. LPS enhanced the glycolysis and polyol pathways. Iron deprivation disrupted the TCA cycle. Iron overload increased intracellular cholesterol. Interestingly, iron deprivation augmented, whereas iron overload diminished, LPS-induced itaconic acid production, which has anti-microbial and anti-inflammatory properties. Therefore, iron-deficient PAMs may be more resistant to intracellular pathogens which use PAMs as a conduit for infection.

Objectives: This study utilized IPEC-J2, a neonatal pig jejunum-derived cell line, to assess how iron deficiency (ID) and excess (IE) alter enterocyte metabolism and the transcription of inflammatory markers. Methods: Cells were treated with deferiprone (DFP) or ferric ammonium citrate (FAC) to induce ID or IE, respectively. The study evaluated: (1) transcriptional changes in iron-regulatory genes over 96 h under ID or IE; (2) the interaction between iron imbalance and lipopolysaccharide (LPS) exposure on mRNA expression of inflammation markers and iron transporters; and (3) cellular metabolic responses to ID, IE, and iron repletion using untargeted metabolomics. Results: ID triggered dynamic transcriptional changes in iron regulatory genes and suppressed cellular proliferation via impaired DNA replication. IE resulted in a persistent reduction in TFRC expression. LPS increased CYBRD1 (p < 0.001) and IL8 (p = 0.004) and tended to elevate TLR4 and TNF expression (p ≤ 0.07), while iron deficiency upregulated IL8 expression (p < 0.001). ID disrupted the TCA cycle, reduced glucuronic acid synthesis, and elevated glycolysis for energy production, whereas IE increased cholesterol biosynthesis and decreased alpha-tocopherol levels. Repletion of iron partially reversed ID-induced metabolic changes. Conclusions: ID impaired enterocyte proliferation and profoundly disrupted cellular metabolism, whereas IE enhanced cholesterol synthesis and depleted alpha-tocopherol levels. Restoration of cellular metabolism following iron repletion was observed, highlighting the resilience of enterocytes.