Iron Deficiency And Overload Attenuate Inflammatory Response and Modulate Metabolism in Alveolar Macrophage in Vitro

Abstract

ObjectiveThe study aimed to characterize immunometabolic signatures of porcine alveolar macrophage (PAM) associated with cellular iron deficiency (ID) and iron overload (IE). We hypothesized that cellular iron imbalance modulates LPS‐induced inflammatory and metabolic responses in PAM cells.MethodsAlveolar macrophage cells were collected from 6‐week old donor piglets (N= 5‐6) and were cultured in complete media at 37 ˚C with 5% CO2 until attachment. Cells were treated with complete media (control, CON) or complete media supplemented with 500 µM deferiprone (DFP) or 200 µM ferric ammonium citrate (FAC) to induce iron deficiency (ID) or iron excess (IE), respectively. At the end of the 36‐hour treatment, cells were challenged with 100 ng/mL LPS (L) or sterile saline (S) for another 6 hours. Cells were tested for viability using XTT assay or harvested for analysis of gene, protein expression, and untargeted metabolome using RT‐qPCR, western blot, and GC time‐of‐flight MS, respectively. Cell culture media were collected for analysis of inflammatory cytokines (TNFα, IL‐6, and IL‐10) using ELISA kits. Data were analyzed for two‐way ANOVA using PROC MIXED of SAS (v.9.4). Statistical significance was declared when P‐value or FDR is less than 0.05 or 0.1, respectively.ResultsLPS challenge reduced cell viability by 10 – 20% regardless of the cellular iron status (P < 0.001). Ferritin H was markedly increased in IE cells compared to CON or ID cells (P < 0.001), suggesting cellular iron overload. The mRNA expression of transferrin receptor 1 was the highest in ID‐S cells and was significantly higher in ID and CON compared to IE cells, irrespective of the LPS challenge (P < 0.001). LPS significantly increased mRNA expression of divalent metal transporter 1 (DMT1)and zinc transporter (ZIP14) by 10‐300 fold (P< 0.001), while iron treatment had no significant effect. Unsurprisingly, LPS drastically induced (P < 0.001) genes encoding pro‐ (TNFA and IL1B) and anti‐(TGFB1 and IL10) inflammatory cytokines. Unexpectedly, both ID and IE diminished the induction of those genes caused by the LPS challenge (P < 0.05). The response of TNFA gene was mirrored by changes in TNFα concentration in culture media. Twenty‐nine, 14, and 5 metabolites were altered by cellular iron status, LPS challenge, and their interaction (FDR < 0.1), respectively. A total of 28 altered metabolites were mapped in the user database. Interestingly, metabolites involved in the glycolysis and TCA cycle (e.g. glucose‐6‐phosphate, citric acid, and aconitic acid) were most abundant in ID cells, whereas, in comparison with CON, both ID and IE decreased several amino acids in PAM (e.g. glycine, alanine, leucine, and lysine). LPS increased metabolites involved in glycolysis (fructose‐1‐phosphate) and pentose phosphate pathway (ribose‐5‐phosphate and fructose‐6‐phosphate). Cellular iron status modulated changes in itaconic acid, glucose, fumaric acid, and putrescine in response to LPS challenge; ID enhanced the LPS‐induced increase in itaconic acid, a metabolite marker of macrophage activation.ConclusionPresence of iron chelator and iron overexposure altered cellular iron metabolism of PAM cells and its inflammatory and metabolic responses to the invasion of bacterial pathogens.