Bruce S. Zwilling

Expression and localization of hepcidin in macrophages: a role in host defense against tuberculosis

Hepcidin is an antimicrobial peptide produced by the liver in response to inflammatory stimuli and iron overload. Hepcidin regulates iron homeostasis by mediating the degradation of the iron export protein ferroportin 1, thereby inhibiting iron absorption from the small intestine and release of iron from macrophages. Here, we examined the expression of hepcidin in macrophages infected with the intracellular pathogens Mycobacterium avium and Mycobacterium tuberculosis. Stimulation of the mouse RAW264.7 macrophage cell line and mouse bone marrow‐derived macrophages with mycobacteria and IFN‐γ synergistically induced high levels of hepcidin mRNA and protein. Similar results were obtained using the human THP‐1 monocytic cell line. Stimulation of macrophages with the inflammatory cytokines IL‐6 and IL‐β did not induce hepcidin mRNA expression. Iron loading inhibited hepcidin mRNA expression induced by IFN‐γ and M. avium, and iron chelation increased hepcidin mRNA expression. Intracellular protein levels and secretion of hepcidin were determined by a competitive chemiluminescence ELISA. Stimulation of RAW264.7 cells with IFN‐γ and M. tuberculosis induced intracellular expression and secretion of hepcidin. Furthermore, confocal microscopy analyses showed that hepcidin localized to the mycobacteria‐containing phagosomes. As hepcidin has been shown to possess direct antimicrobial activity, we investigated its activity against M. tuberculosis. We found that hepcidin inhibited M. tuberculosis growth in vitro and caused structural damage to the mycobacteria. In summary, our data show for the first time that hepcidin localizes to the phagosome of infected, IFN‐γ‐activated cells and has antimycobacterial activity.

Differential iron transport into phagosomes isolated from the RAW264.7 macrophage cell lines transfected with Nramp1Gly169 or Nramp1Asp169.

The transport of iron by RAW264.7 macrophage cell lines transfected with either Nramp1Gly169 (resistant) or Nramp1Asp169 (susceptible) alleles was assessed. We found no difference between resistant and susceptible cells in the rate of Fe import or export when Fe transport was measured in intact cells. In contrast, the rate of Fe import by latex‐bead phagosomes isolated from resistant cells was more than double the rate by latex‐bead phagosomes from susceptible cells. Similarly, phagosomes isolated from resistant cells that had been pre‐labeled with 55Fe‐citrate before phagocytosis contained up to four times as much Fe as the corresponding phagosomes from susceptible cells. Phagocytosis of Mycobacterium avium was accompanied by an increase in the production of hydroxyl radicals by Nramp1Gly169 ‐transfected macrophages but not by macrophages transfected with the susceptible allele. These results are consistent with the hypothesis that Nramp1 functions to transport Fe into the bacterium‐containing phagosome where it serves as a catalyst for the Haber‐Weiss reaction, which accounts for the increased capacity of these cells to limit mycobacterial growth. J. Leukoc. Biol. 66: 113–119; 1999.

Regulation of Toll-Like Receptor 2 Expression by Macrophages Following Mycobacterium avium Infection

Recent studies have implicated Toll-like receptors (TLR), especially TLR2 and TLR4, as sentinel receptors that signal the interaction of macrophages with bacterial pathogens via a NF-κB-mediated pathway. The regulation of TLR gene expression, however, has not been intensively studied. Here, we report that TLR2 mRNA was induced following infection of murine macrophages with Mycobacterium avium. The changes in TLR2 mRNA correlated with an increase in TLR2 surface expression. Infection with M. avium resulted in a concomitant decrease in TLR4 mRNA. The effect of M. avium infection on TLR2 mRNA appeared to be mediated, in part, by TLR2 because the induction of the mRNA was partially blocked by preincubation of the macrophages with an anti-human TLR2 Ab. In contrast, the effect of LPS stimulation was mediated via TLR4 because infection of macrophages from LPSd mice, which do not express active TLR4, resulted in an increase in TLR2 mRNA, while treatment of macrophages from these mice with LPS failed to induce TLR2 mRNA. Several cytokines, including TNF-α, IL-1α, and GM-CSF, but not IFN-γ, induced TLR2 mRNA. M. avium infection resulted in the induction of TLR2 mRNA by macrophages from both TNFRI knockout and NF-κB p50 knockout mice.

NFκB and Sp1 Elements Are Necessary for Maximal Transcription of Toll-like Receptor 2 Induced by Mycobacterium avium

We have previously reported that Toll-like receptor (TLR) 2 mRNA was induced after infection with Mycobacterium avium. To investigate the molecular basis of TLR2 expression in macrophages, we cloned and analyzed the murine putative 5′-proximal promoter. Transient transfection of a 326-bp region from nucleotides −294-+32 relative to the first transcription start site was sufficient to induce maximal luciferase activity at the basal level and after infection with M. avium in J774A.1 cells. Sequence analysis showed that the region lacked a TATA box but contained two typical stimulating factor (Sp) 1 sites, two NF-κB sites, one IFN-regulatory factor site and one AP-1 site. Site-directed mutagenesis revealed that the NF-κB and Sp1 sites but not the IFN-regulatory factor site or the AP-1 site contributed to the basal level and the induction of luciferase activity during M. avium infection. Binding of Sp1/Sp3 and NF-κB (p50/p65) was confirmed by EMSA. Further studies showed that three copies of Sp1 elements or NF-κB elements are not sufficient to confer M. avium induction on a heterologous promoter. By contrast, overexpression of NF-κB p65 caused a strong increase in transcription from an intact TLR2 promoter, whereas it caused only a partial increase in promoter activity when cotransfected with the TLR2 promoter with one of the Sp1 sites mutated. Sp1 and NF-κB were the minimum mammalian transcription factors required for effective TLR2 transcriptional activity when transfected into Drosophila Schneider cells. Together, these data provide genetic and biochemical evidence for NF-κB as well as Sp1 in regulating TLR2 transcription.

Mycobacteria Inhibition of IFN-γ Induced HLA-DR Gene Expression by Up-Regulating Histone Deacetylation at the Promoter Region in Human THP-1 Monocytic Cells

Infection of macrophages with mycobacteria has been shown to inhibit the macrophage response to IFN-γ. In the current study, we examined the effect of Mycobacteria avium, Mycobacteria tuberculosis, and TLR2 stimulation on IFN-γ-induced gene expression in human PMA-differentiated THP-1 monocytic cells. Mycobacterial infection inhibited IFN-γ-induced expression of HLA-DRα and HLA-DRβ mRNA and partially inhibited CIITA expression but did not affect expression of IFN regulatory factor-1 mRNA. To determine whether inhibition of histone deacetylase (HDAC) activity could rescue HLA-DR gene expression, butyric acid and MS-275, inhibitors of HDAC activity, were added at the time of M. avium or M. tuberculosis infection or TLR2 stimulation. HDAC inhibition restored the ability of these cells to express HLA-DRα and HLA-DRβ mRNA in response to IFN-γ. Histone acetylation induced by IFN-γ at the HLA-DRα promoter was repressed upon mycobacteria infection or TLR2 stimulation. HDAC gene expression was not affected by mycobacterial infection. However, mycobacterial infection or TLR2 stimulation up-regulated expression of mammalian Sin3A, a corepressor that is required for MHC class II repression by HDAC. Furthermore, we show that the mammalian Sin3A corepressor is associated with the HLA-DRα promoter in M. avium-infected THP-1 cells stimulated with IFN-γ. Thus, mycobacterial infection of human THP-1 cells specifically inhibits HLA-DR gene expression by a novel pathway that involves HDAC complex formation at the HLA-DR promoter, resulting in histone deacetylation and gene silencing.

Cloning and characterization of a novel cDNA that is IFN-gamma-induced in mouse peritoneal macrophages and encodes a putative GTP-binding protein.

Macrophage activation by IFN‐γ results in a cascade of gene expression. To identify genes activated in mouse peritoneal macrophages by IFN‐γ, we created a cDNA subtraction library of IFN‐γ‐induced genes. We have isolated from this subtraction library a novel cDNA clone, called Mg21, whose mRNA is absent in unstimulated mouse peritoneal macrophages and is induced to high levels within 4 h following the addition of IFN‐γ. Induction of Mg21 mRNA by IFN‐γ occurred in the presence of cycloheximide, indicating that expression of Mg21 mRNA does not require protein synthesis. A small amount of Mg21 mRNA was also induced by LPS, but not by IL‐2, IL‐4, IL‐10, or TNF‐α. The DNA sequence of Mg21 is 1617 nucleotides and contains an open reading frame that codes for a protein of 415 amino acids with a predicted molecular weight of 47,106 Da. The predicted amino acid sequence lacks a signal sequence or transmembrane segments, indicating that the protein is an intracellular protein. Computer search of the GenBank and EMBL databases indicates that this cDNA clone is unique but has 57% sequence identity with IRG‐47, which is a mouse gene induced by IFN‐γ in pre‐B and B lymphocyte cell lines. IRG‐47 encodes an intracellular protein that contains three conserved protein motifs present in GTP‐binding proteins. Analysis of the protein sequence of Mg21 showed that these three conserved protein motifs are also present in Mg21. J. Leukoc. Biol. 57: 477–483; 1995.

Iron transport into Mycobacterium avium‐containing phagosomes from an Nramp1Gly169‐transfected RAW264.7 macrophage cell line

Nramp1 is an important determinant of innate resistance of macrophages to the growth of intracellular microorganisms. We previously showed that Nramp1 functions to transport iron from the cytoplasm into phagosomes of Mycobacterium avium‐infected macrophages. The purpose of this investigation was to further characterize the factors that regulate Nramp1‐mediated iron transport into phagosomes. Treatment of Nramp1Gly169 macrophages with the lysomotrophic agents chloroquine or ammonium chloride reduced the import of iron significantly. We found that macrophage‐activating cytokines, including TNF‐α, IFN‐γ, IL‐1α, and GM‐CSF, when added prior to M. avium, increased the transport of iron into the phagosome. This increase in iron transport was not a result of an increased amount of Nramp1 protein in the phagosome nor to new protein synthesis. Treatment of Nramp1Gly169‐transfected macrophages with inhibitors of protein kinase C (PKC) diminished the import of iron into the phagosomes. Iron import was inhibited by an anti‐Nramp1 antibody against the putative fourth outer‐loop region of Nramp1 but not by an anti‐Nramp1 antibody against the carboxy terminus. The significance of these results on the orientation of Nramp1 in the phagosome membrane and on the transport of iron is discussed.

Role of STAT1, NF-κB, and C/EBPβ in the macrophage transcriptional regulation of hepcidin by mycobacterial infection and IFN-γ

Hepcidin is an antimicrobial peptide involved in regulating iron homeostasis. It is induced by iron overload and decreased by hypoxia and anemia. Hepcidin regulates iron metabolism by inhibiting iron absorption by the duodenum and by inhibiting macrophage iron recycling. Hepcidin is induced in hepatocytes during the acute‐phase response by IL‐6. Previously, we have shown that hepcidin is not induced in macrophages by IL‐6 but is induced by the synergistic interaction of IFN‐γ and Mycobacterium tuberculosis infection. In the present study, we examined the pathways involved in inducing macrophage hepcidin expression. We show that TLRs TLR2 and TLR4 and the transcription factor STAT1 are required for induction of hepcidin mRNA. Hepcidin promoter activity is also synergistically induced in RAW264.7 macrophages by IFN‐γ and M. tuberculosis. NF‐κB and C/CEBP binding sites are required for promoter activity. Binding of NF‐κB (p50/p65) to the NF‐κB site and STAT1 and C/EBPβ to the C/CEBP site was confirmed by EMSA. Knockdown of STAT1 and C/EBPβ expression in RAW264.7 cells with siRNA plasmids inhibited hepcidin promoter activity induced by IFN‐γ and M. tuberculosis. Together, these studies demonstrate that macrophage hepcidin expression is induced by the activation of STAT1 and NF‐κB and the induction of C/EBPβ expression.

Activation of the hypothalamic-pituitary-adrenal axis differentially affects the anti-mycobacterial activity of macrophages from BCG-resistant and susceptible mice

The effect of hypothalamic-pituitary-adrenal (HPA) axis activation and exogenous glucocorticoids on the ability of splenic macrophages to control the growth of Mycobacterium avium was evaluated. We found that activation of the HPA axis by restraint stress or the addition of corticosterone increased the susceptibility of macrophages from mice that are innately susceptible to the in vivo growth of M. avium. In contrast, the ability of macrophages from innately resistant, congenic mice to control the growth of M. avium was not affected by HPA activation or the addition of corticosterone. The effect of restraint and of corticosterone on macrophage function was abrogated by either treating mice with the glucocorticoid receptor antagonist RU486 or the addition of the drug to cultures of macrophages. Activation of the HPA axis as well as the addition of corticosterone to cultures of macrophages resulted in a suppression of the production of tumor necrosis factor (TNF)-alpha and of reactive nitrogen intermediates by macrophages from both strains of mice. The lack of effect of HPA activation and of corticosterone on the mycobacterial resistance of macrophages from BCG-resistant mice, while at the same time suppressing the production of reactive nitrogen intermediates, appears to rule out a role for this antimicrobial pathway in innate resistance to mycobacterial growth.

The iron export protein ferroportin 1 is differentially expressed in mouse macrophage populations and is present in the mycobacterial-containing phagosome

Intracellular pathogens, including Mycobacterium tuberculosis, obtain iron from the host for their survival. Ferroportin 1 (FPN1; SLC40A1) is the sole iron exporter from mammalian cells and is expressed in the duodenum and macrophages. In the present study, we show that FPN1 mRNA levels in the mouse macrophage cell line RAW264.7 are synergistically induced by treatment with live or γ‐irradiated M. tuberculosis and IFN‐γ. FPN1 mRNA levels were also induced by Mycobacterium avium and IFN‐γ in RAW264.7 cells and the mouse alveolar macrophage cell line AMJ2‐C8. Treatment of mouse resident peritoneal macrophages with M. tuberculosis and IFN‐γ resulted in a sixfold increase in FPN1 mRNA expression. In contrast, M. tuberculosis and IFN‐γ inhibited FPN1 mRNA expression in bone marrow‐derived macrophages and lung macrophages, which have high basal levels of FPN1 mRNA expression. Using confocal microscopy, FPN1 protein localized rapidly to M. tuberculosis phagosomes after infection in RAW264.7 macrophages. In RAW264.7 cells expressing wild‐type natural resistance‐associated macrophage protein 1 (Nramp1Gly169), FPN1 and Nramp1 partially colocalized in late endosomes/lysosomes prior to infection. After 2 h of infection, Nramp1 and FPN1 were present in M. tuberculosis phagosomes. Our studies provide evidence for transcriptional regulation of FPN1 by pathogenic mycobacteria and IFN‐γ, which is dependent on the macrophage type. The trafficking of FPN1 to the M. tuberculosis phagosome suggests that it is involved in regulating iron availability to the mycobacteria in this locale.