Cell iron status influences macrophage polarization

Abstract

Macrophages play crucial roles in innate immune response and in the priming of adaptive immunity, and are characterized by their phenotypic heterogeneity and plasticity. Reprogramming intracellular metabolism in response to microenvironmental signals is required for M1/M2 macrophage polarization and function. Here we assessed the influence of iron on the polarization of the immune response in vivo and in vitro. Iron-enriched diet increased M2 marker Arg1 and Ym1 expression in liver and peritoneal macrophages, while iron deficiency decreased Arg1 expression. Under LPS-induced inflammatory conditions, low iron diet exacerbated the proinflammatory response, while the IL-12/IL-10 balance decreased with iron-rich diet, thus polarizing toward type 2 response. Indeed, in vitro macrophage iron loading reduced the basal percentage of cells expressing M1 co-stimulatory CD86 and MHC-II molecules. Further, iron loading of macrophages prevented the pro-inflammatory response induced by LPS through reduction of NF-κB p65 nuclear translocation with decreased iNOS, IL-1β, IL-6, IL-12 and TNFα expression. The increase of intracellular iron also reduced LPS-induced hepcidin gene expression and abolished ferroportin down-regulation in macrophages, in line with macrophage polarization. Thus, iron modulates the inflammatory response outcome, as elevated iron levels increased M2 phenotype and negatively regulated M1 proinflammatory LPS-induced response.

Fig 1. Association of iron status and immune response marker expression in liver and peritoneal macrophages.

C57BL/6 wild type mice were fed with iron replete diet (IR), deficient diet for 14 days (Low) or iron-rich diet for 3 days (High). (A) Body weight and relative liver weights are indicated. Liver homogenates (B, C), and peritoneal macrophages (D) gene expression was then analyzed using quantitative PCR analysis. Data are expressed as mRNA fold change relative to control mice fed with IR diet. Data are from two independent experiments and presented as mean ± SD (n = 6 mice per group). ns, non significant; * p≤ 0.05; ** p≤ 0.01; *** p≤ 0.001.

Fig 2. Iron status in liver, spleen and peritoneal macrophages.

C57BL/6 wild type mice were fed with iron replete diet (IR), or iron-rich diet for 3 days (Fe diet) or received an injection of iron-dextran (Fe-dextran, 0.2 g/kg ip) for 48h. (A) Body weight, relative liver and spleen weights are indicated. (B) Representative DAB-enhanced Perl’s staining of liver, spleen, and peritoneal exudate cells spin show iron deposit (in brown). (C) Hepcidin 1 (Hamp1) and ferritin L (Ftl) gene expression in liver tissue homogenates, spleen and peritoneal cells was analyzed using quantitative PCR. Data are expressed as mRNA fold change relative to control mice fed with IR diet. Data are from two independent experiments and presented as mean ± SD (n = 6 mice per group). ns, non significant; * p≤ 0.05; ** p≤ 0.01; *** p≤ 0.001.

Fig 3. Expression of macrophage polarization markers upon iron overload in liver, spleen and peritoneal macrophages.

C57BL/6 wild type mice were fed with iron replete diet (IR), or iron-rich diet for 3 days (Fe diet) or received an injection of iron-dextran (Fe-dextran, 0.2 g/kg ip) for 48h. Liver homogenates (A), spleen (B) and peritoneal exudate macrophages (C) gene expression of macrophage polarization marker Arginase-1 (Arg1), Chitinase-like 3 (Ym1), CD206 mannose receptor (Mrc) or iNos was analyzed using quantitative PCR analysis. Data are expressed as mRNA fold change relative to control mice fed with IR diet. Data are from two independent experiments and presented as mean ± SD (n = 6 mice per group). ns, non significant; * p≤ 0.05; ** p≤ 0.01; *** p≤ 0.001.

Fig 4. Iron deficiency increases LPS-induced IL-12/IL-10 balance in vivo.

C57BL/6 wild type mice were either fed with iron replete diet (IR) or iron-deficient diet for 14 days (Low) or iron-rich diet for 3 days (High), or treated with iron-dextran (Fe-dextran, 0.2 g/kg ip) for 48h. Mice were then treated with LPS (i.p 50 μg/kg) or NaCl 0.9% as vehicle for 4 h. Peritoneal cells were collected to assess the Il-12, Il-10, and Il-1β gene expression using quantitative PCR. (A) data from mice fed with various diets and (B) comparison of mice fed with high iron diet or treated with iron-dextran. Data are expressed as mRNA fold change relative to vehicle control mice under IR diet. Gene expression was then analyzed using quantitative PCR analysis. Data are expressed as mRNA fold change relative to control mice fed with IR diet. Data are from two independent experiments and presented as mean ± SD (n = 6 mice per group). ns, non significant; ** p≤ 0.01; *** p≤ 0.001.

Fig 5. Iron loaded macrophages express constitutive M1 cell surface markers in vitro.

A: Bone marrow-derived macrophages incubated in 6-wells plate were treated overnight with ferric ammonium citrate (FAC 0, 50 or 100 μM) as indicated. Thereafter, cells were stained by 7-Aminoactinomycin D (7-AAD) and membrane integrity analyzed by flow cytometry. The percentage of viable cells is given by excluding all the positive cells. B: Bone marrow-derived macrophages plated in 96 well microplate overnight were washed with PBS and treated with either calcein-AM (5 μM, 5 min) for labile cell iron measurement or with H2DCFDA (5 μM, 30 min) for intracellular reactive oxygen species (ROS) assessment. After addition of ferric ammonium citrate (FAC, 10, 50 or 100 μM), fluorescence measurements were performed at different time points to determine the level of labile cell iron or after 16 hours for intracellular ROS. C: Bone marrow-derived macrophages incubated in 6-wells plate were preincubated 2 hours with ferric ammonium citrate (FAC, 0, 50 or 100 μM), then incubated with IL-4 (10 ng/mL) or IFNγ (100 UmL) overnight. The percentage of CD86⁺, I-A/I-E⁺ and CD206⁺ cells were then assessed in F4/80⁺ cells. Data are representative of two independent experiments and presented as mean ± SD (n = 4). * p≤ 0.05; ** p≤ 0.01; *** p≤ 0.001.

Fig 6. Iron loading of primary macrophages impairs LPS-induced pro-inflammatory responses.

Bone marrow derived-macrophages were preincubated with ferric ammonium citrate (FAC, 0, 50 or 100 μM) overnight followed by LPS stimulation (100 ng/mL) for 4 h or 24 h to assess Il-12b, Il-1β, Il-6, Tnfα and iNos mRNA gene expression by quantitative PCR analysis (A) or cytokine Il-12p40, Il-1β, Il-6, Tnfα and nitric oxide measurement in cell medium by ELISA and Griess assay (B), respectively. Bone marrow derived-macrophages were preincubated with ferric ammonium citrate (FAC 100 μM) or medium overnight followed by LPS stimulation (100 ng/mL) for 30 min to assess NF-κB nuclear translocation by immunofluorescence, with NF-κB p65 in green and DAPI in red (C). Data of mRNA gene expression are given as fold change gene expression relative to the expression in untreated cells. Data are representative of at least two independent experiments and presented as mean ± SD (n = 4). * p≤ 0.05; ** p≤ 0.01; *** p≤ 0.001.

Fig 7. Iron loading modulates LPS-induced hepcidin 1 and ferroportin expression in macrophages.

Bone marrow derived-macrophages were incubated with ferric ammonium citrate (FAC, 0, 50 or 100 μM) for 16 hours followed by LPS stimulation (10 ng/mL). Hepcidin 1 (Hamp1) and ferroportin (Fpn) mRNA expression were determined after 4 h (Fpn) or 12 h (Hamp1) using quantitative PCR analysis. Data are presented as fold change gene expression relative to the expression in untreated cells. Data are representative of at least three independent experiments and presented as mean ± SD (n = 4). *** p≤ 0.001.