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-gamma 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-gamma and M. tuberculosis. NF-kappaB and C/CEBP binding sites are required for promoter activity. Binding of NF-kappaB (p50/p65) to the NF-kappaB site and STAT1 and C/EBPbeta to the C/CEBP site was confirmed by EMSA. Knockdown of STAT1 and C/EBPbeta expression in RAW264.7 cells with siRNA plasmids inhibited hepcidin promoter activity induced by IFN-gamma and M. tuberculosis. Together, these studies demonstrate that macrophage hepcidin expression is induced by the activation of STAT1 and NF-kappaB and the induction of C/EBPbeta expression.
Mycobacterial infections of macrophages have been shown to inhibit the ability of the macrophage to respond to IFN-gamma. We previously reported that Mycobacterium avium infection of mouse macrophages decreases IFN-gamma-induced STAT1 tyrosine phosphorylation and STAT1 DNA binding. Because macrophages respond to M. avium through Toll-like receptor 2 (TLR2), we determined whether TLR2 stimulation inhibits the response to IFN-gamma. Treatment of mouse RAW264.7 macrophages with TLR2 agonists inhibited the induction of IFN-gamma-inducible genes by IFN-gamma. In contrast to M. avium infection, TLR2 agonists did not inhibit the IFN-gamma induction of DNA-binding activity of STAT1 and the tyrosine phosphorylation of STAT1alpha. Instead, IFN-gamma induction of RAW264.7 cells treated with TLR2 agonists resulted in an increase in the tyrosine phosphorylation of the dominant-negative STAT1beta. TLR2 stimulation of RAW264.7 cells increased both STAT1beta protein and mRNA expression, suggesting that the increased STAT1beta phosphorylation results from increased STAT1beta expression. Because STAT1alpha and STAT1beta mRNA have different 3' untranslated regions, and 3' untranslated regions can regulate mRNA stability, we examined the effects of TLR2 stimulation on mRNA stability. TLR2 stimulation of RAW264.7 cells increased the stability of STAT1beta mRNA, while not affecting the stability of STAT1alpha mRNA. The ability of STAT1beta to function as a dominant negative was confirmed by overexpression of STAT1beta in RAW264.7 macrophages by transient transfection, which inhibited IFN-gamma-induced gene expression. These findings suggest that M. avium infection of mouse macrophages inhibits IFN-gamma signaling through a TLR2-dependent increase in STAT1beta expression by mRNA stablization and a TLR2-independent inhibition of STAT1 tyrosine phosphorylation.
Interferon-gamma (IFN-gamma) is a key cytokine in the immune defense against mycobacteria. IFN-gamma activates macrophages to resist the growth of mycobacteria and induces expression of MHC class II molecules required for antigen presentation. Macrophages infected with mycobacteria or stimulated by the interaction of mycobacterial products with toll-like receptor 2 (TLR2) have reduced responses to IFN-gamma. Previous research has shown that infection of mouse macrophages with Mycobacterium avium causes decreased expression of the IFN-gamma receptor (IFNGR). In the present study, we show that TLR2 stimulation of RAW264.7 macrophages with a synthetic lipoprotein, Pam3CSK4, also causes rapid decrease in expression of IFNGR-1 protein, with little change in IFNGR-2 protein levels. The decrease in IFNGR-2 expression in TLR2-stimulated cells required receptor internalization and proteasomal degradation. The level of IFNGR-1 mRNA also decreased in TLR2-stimulated RAW264.7 cells and M. avium-infected cells. The decrease in IFNGR-1 mRNA was shown to be due to decreased transcription. In spite of the decrease in IFNGR-2 receptor expression, activation of Stat1 activation by an optimal dose of IFN-gamma was identical between control and TLR2-stimulated RAW264.7 cells. However, at low suboptimal doses of IFN-gamma, Stat1 activation was decreased in TLR2-stimulated cells.
Mycobacteria-infected macrophages are poor responders to interferon-gamma (IFN-gamma), resulting in decreased expression of IFN-gamma-induced genes. In the present study, we examined the inhibition of IFN-gamma-induced gene expression by Mycobacterium tuberculosis and four different Mycobacterium avium strains in mouse RAW264.7 macrophages. Gamma-irradiated M. tuberculosis inhibited mRNA expression of a panel of six different IFN- gamma-induced genes. All four of the M. avium strains completely inhibited IFN-gamma-induced expression of MHC class II Aalpha and Ebeta mRNA. However, the Mac101 strain, which is serovar 1, inhibited IFN-gamma induction of IFN regulatory factor-1 (IRF-1) and guanylate-binding protein-1 (GBP-1) mRNA to a greater extent than the other M. avium strains, which are serovar 2. In this study, we also show that mycobacteria inhibit gene expression by both toll-like receptor 2 (TLR2)-dependent and independent pathways. The inhibition of IFN-gamma-induced gene expression by M. avium was reduced but not completely blocked in macrophages from TLR2(/) mice. IFN-gamma-induced gene expression was also inhibited by mycobacteria in RAW264.7 cells expressing dominantnegative TLR2 or myeloid differentiation factor 88 (MyD88), further indicating the existence of a pathway independent of TLR2 and MyD88. These data suggest that mycobacteria inhibit IFN-gamma-induced gene expression by multiple pathways involving both TLR2 and non-TLR receptors.
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