Edited by: Celio Geraldo Freire De Lima, Federal University of Rio de Janeiro, Brazil
Reviewed by: Mireya De La Garza, Center for Research and Advanced Studies of the National Polytechnic Institute (CINVESTAV), Mexico; Mary Fafutis-Morris, University of Guadalajara, Mexico
Specialty section: This article was submitted to Microbial Immunology, a section of the journal Frontiers in Immunology
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Human lactoferrin (hLf), an 80-kDa multifunctional iron-binding cationic glycoprotein, is constitutively secreted by exocrine glands and by neutrophils during inflammation. hLf is recognized as a key element in the host immune defense system. The
Iron is essential for living organisms, being required in many proteins for a broad range of vital functions, such as oxygen transport and energy production. However, it can be toxic when in excess due to its ability to partially reduce oxygen, thus generating reactive oxygen species, which are known to cause tissue injury and organ failure by damaging a number of cellular components, including DNA, proteins, and membrane lipids. This has led to the evolution of strictly controlled pathways of iron uptake and release to minimize its deficiency or excess (
Systemic iron homeostasis is tightly controlled in humans. Both iron acquisition by enterocytes and iron recycling by macrophages are regulated through iron absorption, storage, and export, as mammals lack a direct iron excretion system (
Dietary iron enters the body at the duodenal level through the concerted action of the ferrireductase DCYTB and the metal importer DMT-1 (
During infection and inflammation, iron homeostasis is grossly perturbed, leading to iron disorders. Enterocytes and macrophages become iron overloaded, thus increasing the host susceptibility to infections (
In this respect, specific cells express molecules that sequester iron to promote host defense. In particular, many mucosal epithelia secrete human lactoferrin (hLf), an 80-kDa iron-binding cationic glycoprotein, which, at variance with transferrin, is able to chelate two Fe (III) per molecule with high affinity even at the very low pH values, characteristic of inflamed and infected sites (
In addition to the antimicrobial properties dependent and independent from its iron-binding ability, hLf exhibits a variety of effects on the host immune system, ranging from inhibition of inflammation to promotion of both innate and adaptive immune responses (
Bovine Lf (bLf), which shares high sequence homology with the human protein, is also a multifunctional glycoprotein with identical antibacterial, antifungal, antiviral, antiparasitic (
Evidence indicates that bLf modulates inflammation by affecting expression of cytokines, chemokines, and other effector molecules. For instance, oral administration of bLf modulates the expression of IL-6, the main cytokine involved in inflammatory and iron homeostasis, reverting homeostasis disorders in pregnant women suffering from IDA and AI (
Previously, we demonstrated that bLf affects iron homeostasis in inflamed models of both epithelial and macrophagic cells by inhibiting IL-6 production and rescuing the expression of the iron exporter Fpn (
Although the mechanisms underlying bLf anti-inflammatory properties have not been fully elucidated yet, its interaction with macrophages may play a critical role.
In this work, we have extended our study, investigating the effect of bLf on the expression of all pivotal proteins (Fpn, membrane-bound Cp, cytosolic Ftn, transferrin receptor 1, and cytokines) involved in mammalian iron and inflammatory homeostasis in a model of inflamed human macrophagic THP-1 cells, challenged with a mixture of LPS and IFN-γ or with LPS alone. The rationale is that macrophages can adapt their phenotype in response to different environmental stimuli and that the iron system proteins are expressed at different extent in different macrophagic phenotypes. Here, we reported the effect of bLf added both to a classical mixture of low concentrations of LPS and IFN-γ, known to induce a pure M1 polarization, and to high concentrations of LPS, known to induce a mixed inflammatory M1/tolerogenic M2 phenotypic population on the expression of iron and inflammatory homeostasis.
Highly purified bLf was kindly provided by Morinaga Milk Industries Co., Ltd. (Tokyo, Japan). The purity of bLf was checked by SDS-PAGE and silver nitrate staining, while its concentration was assessed by UV spectroscopy on the basis of an extinction coefficient of 15.1 (280 nm, 1% solution). The bLf iron saturation was about 20% as detected by optical spectroscopy at 468 nm on the basis of an extinction coefficient of 0.54 (100% iron saturation, 1% solution). LPS contamination of bLf, estimated by Limulus Amebocyte assay (Pyrochrome kit, PBI International), was equal to 0.7 ± 0.06 ng/mg of bLf. Before biological assays, bLf solution was sterilized by filtration using 0.2 µm Millex HV at low protein retention (Millipore Corp., Bedford, Mass.). In all experiments, bLf was used at a non-cytotoxic concentration corresponding to 100 µg/ml.
THP-1 cells, a myelomonocytic cell line derived from the blood of a 1-year-old boy with acute monocytic leukemia (ECACC, European Collection of Cell Cultures), were maintained in RPMI 1640 medium (Euroclone, Italy), supplemented with 10% fetal calf serum and 2 mM glutamine, in an atmosphere of 95% air and 5% CO2. Cells, which grow spontaneously in loose suspension under these conditions, were subcultured twice a week by gentle shaking followed by pelleting and reseeding at a density of approximately 106 cells/ml.
THP-1 cells were differentiated in macrophages by incubation in 25 cm2 flasks at a density of approximately 106 cells/ml in 5 ml of RPMI (supplemented with 100 µM penicillin–streptomycin, 2 mM glutamine, 10% fetal calf serum) containing 0.16 µM phorbol myristate acetate (PMA, Sigma Chemical Co.) for 48 h, at 37°C in an atmosphere of 95% air and 5% CO2 (
Quantitation of IL-6, IL-1β, and IL-10 was performed on cell monolayer supernatants by ELISA, using Human ELISA Max Deluxe Sets (BioLegend, USA).
THP-1 cells (about 5 × 106 cells) were lysed in 300 µl of lysis buffer (25 mM 3-morpholinopropane-1-sulfonic acid pH 7.4/150 mM NaCl/1% Triton containing 1 mM PMSF, 2 µM leupeptin, and pepstatin) in ice for 1 h. Total protein content of samples was measured by Bradford assay. For SDS-PAGE, 20 µg of total protein in SDS sample buffer containing 1,4-dithiothreitol, were heat-treated (except for Fpn) and loaded. For Western Blot analysis, primary antibodies used were: monoclonal anti-Fpn 31A5, generously provided by T. Arvedson (Amgen) (1:10,000) (
All experiments were run at least in triplicate. Results are expressed as mean ± SE. Statistical analysis was performed with GraphPad Prism and analysis of variance was used to compare quantitative data populations with normal distribution and equal variance.
Preliminary experiments showed that THP-1 cells are usually very prone to rapid aging. As shown in Figure
Increase of IL-6 levels in THP-1 cells, differentiated in macrophages by phorbol myristate acetate treatment, cultured for different times. T15–T60 refer to days after the cell batch had been thawed.
To induce the inflammatory macrophagic phenotype M1, cells were stimulated with 10 pg/ml LPS and 20 ng/ml IFN-γ, alone or in combination, in the presence or absence of 100 µg/ml bLf.
As shown in Figures
Changes in IL-6
Ferroportin and Cp-GPI synthesis followed the same pattern, with expression of both proteins severely impaired when cells were challenged with the mixture of LPS and IFN-γ (Figures
Expression of TfR1 and Ftn was also found to change, and again production of the relative proteins was significantly affected only by the combined treatment with both LPS and IFN-γ, while being essentially unchanged after single stimulatory treatments. However, TfR1 expression was ca. 40% downregulated by the mixture (Figure
When IL-10 was measured as a marker of the tolerogenic, anti-inflammatory status of macrophages, it turned out that the treatment with the LPS/IFN-γ mixture dramatically reduced the cytokine levels (Figure
Changes in IL-10 levels in THP-1 cells stimulated with
In order to assess whether the observed effects were confined to pro-inflammatory macrophages induced by the LPS/IFN-γ mixture, we treated differentiated THP-1 with high concentrations of LPS alone. Under these conditions, macrophages likely evolve into a heterogeneous population with phenotypes ranging from pro-inflammatory M1 to tolerogenic M2 (
Changes in IL-6
As with the stimulation with the LPS/IFN-γ mixture, no effect on IL-6, IL-1β, Fpn, TfR1, Ftn, and Cp-GPI production was detected in the control cells treated with bLf alone.
The changes of IL-10 observed upon stimulation with 1 µg/ml LPS were at odd with those observed with the LPS/IFN-γ mixture. As shown in Figure
Representative Western Blot assays on Fpn, TfR1, Ftn, and Cp-GPI in THP-1 cells stimulated both with a mixture of 20 ng/ml IFN-γ and 10 pg/ml LPS or with LPS 1 µg/ml, in the presence or absence of 100 µg/ml bLf are shown in Figure
Representative Western Blot assays on ferroportin (Fpn), membrane-bound ceruloplasmin (Cp), transferrin receptor 1, and cytosolic ferritin (Ftn) in THP-1 cells stimulated both with a mixture of 20 ng/ml IFN-γ and 10 pg/ml LPS or with LPS 1 µg/ml, in the presence or absence of 100 µg/ml bovine Lf (bLf).
Microorganisms require iron for the biosynthesis of macromolecules, thus pathogens have developed efficient systems to take up iron from the host. In turn, hosts rely on innate immune strategies that limit iron availability by finely regulating host genes involved in iron homeostasis. In this respect, it is of utmost importance to analyze how macrophages, which are crucial cellular types of the innate immune system, respond to inflammatory stimuli. Our data clearly show that a whole set of proteins involved in iron homeostasis is coordinately regulated upon stimulation of macrophages with inflammatory triggers. These changes take part into intracellular iron overload, a very unsafe condition
Here, we clearly demonstrate that the anti-inflammatory effect of bovine lactoferrin is exerted on all examined components of the iron homeostasis machinery. It should be reminded that macrophages are a heterogeneous population of immune cells resulting from cytokine stimulation and pathogen or its products sensing. Indeed, depending on the microenvironment, macrophages are able to polarize into active subpopulations, with a continuum of macrophage subsets ranging from pro-inflammatory M1 to regulatory/anti-inflammatory M2 phenotypes. Stimulation of macrophages with low-dose LPS and IFN-γ leads to classical pro-inflammatory M1 polarization, while the widely used high-dose LPS stimulus gives rise to a more heterogeneous phenotype (
In this work, we demonstrate that bovine lactoferrin affects the expression of Fpn, TfR1, Ftn, and Cp-GPI in “pure” M1 macrophages obtained by stimulation with low-dose LPS and IFN-γ as well as in a more heterogeneous macrophage population, challenged with high-dose LPS.
In line with the expected “iron-retention” phenotype, levels of Ftn are increased while TfR1, Fpn, and Cp-GPI are decreased in M1 macrophages (low-dose LPS and IFN-γ). We demonstrate that bovine lactoferrin efficiently counteracts the effect of LPS and IFN-γ, restoring Fpn, Cp-GPI, Ftn, and TfR1 levels to those of unstimulated, uninflamed cells. The decrease of the anti-inflammatory cytokine IL-10 induced by low-dose LPS and IFN-γ is also counteracted by bovine lactoferrin.
Also in the case of stimulation with high-dose LPS, the phenotype appears to be one of “iron-retention” with higher levels of Ftn and lower expression of iron uptake/export proteins TfR1, Fpn, and Cp-GPI. Cytokines IL-6 and IL-1β are expressed at much higher levels compared to low-dose LPS/IFN-γ, indicating strong inflammatory conditions. Again, bovine lactoferrin dampens these effects, reducing the changes observed in its absence. In this case, however, levels of IL-10 significantly increase upon LPS stimulation [in line with other reports, e.g., Ref. (
It is interesting to note that bovine lactoferrin exerts its effect both under conditions mimicking the initial stages of infection (i.e., treatment with high doses of sole LPS) and when an inflammatory outcome has been set up (i.e., treatment with low doses of LPS and with IFN-γ). This suggests that, within this experimental framework, bovine lactoferrin acts as an anti-inflammatory agent able to both prevent the onset of inflammation and to relieve it once it has been established.
A last comment deserves to be made regarding the effect of inflammatory stimuli on Cp. Cp is well known to be a positive acute-phase reactant, i.e., a protein upregulated in the systemic response which usually follows a physiological condition that takes place in the beginning of an inflammatory process. Thus, Cp levels are greatly increased in plasma under inflammatory conditions (
Overall, the capacity of bovine lactoferrin to reduce pro-inflammatory cytokine production and to prevent the changes of the whole set of proteins involved in iron homeostasis, in inflamed macrophages, underlines the pivotal role of this natural compound in the complex orchestration of iron and inflammatory homeostasis. Several drugs capable of modulating macrophagic phenotypes are emerging as attractive molecules for treating inflammation, and in this sense, bovine lactoferrin is no exception.
PV, MP, FB, and GM conceived and designed the experiments; AC, LR, ML, and MS performed the experiments and analyzed the data; PV and GM coordinated data collection and data quality assurance; AC, PV, and GM wrote the first draft of the manuscript. All authors read and approved the final version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.