Abstract
Abstract 2111
Pathogenic bacteria must acquire iron from their hosts to survive and have evolved multiple mechanisms to capture iron or iron-containing heme from the bloodstream or tissues. In response, mammals have developed defense mechanisms to keep iron from pathogens. For example, in response to inflammatory cytokines, hepcidin secreted by the liver binds to the iron exporter ferroportin (FPN1), leading to FPN1 internalization and degradation, decreasing gastrointestinal iron absorption and increasing macrophage iron storage. Much of the body's iron stores are complexed in heme. The Feline leukemia virus, subgroup C (FeLV-C) receptor, FLVCR, is a heme export protein. We showed previously that FLVCR is required for the normal development of the erythroid [Science (2008)319:825] and T cell lineages [Blood (ASH Annual Meeting Abstracts)114:913,2009].
Although macrophages express high levels of FLVCR, the role of FLVCR in regulating heme-iron after infection remains unexplored. Other heme regulatory proteins, such as heme oxygenase-1 (HMOX1), a heme-degrading enzyme, are known to be transcriptionally regulated in macrophages in response to infection. We hypothesized that macrophages dynamically regulate Flvcr in response to bacterial infection. To test this hypothesis, we stimulated J774, a murine macrophage cell line, with lipopolysaccharide (LPS from E. coli O111:B4) at varying concentrations and durations. LPS, an outer membrane component from gram-negative bacteria, binds to Toll-like receptor 4 (TLR4) on macrophages and activates downstream signaling pathways. Using multiplex quantitative reverse transcription polymerase chain reaction (qRT-PCR), we measured mRNA levels of Flvcr, Hmox1, and Fpn1. We found that J774 cells down-regulated Flvcr transcript levels in response to LPS with a maximal decrease (69%) seen at 6–8 hours of stimulation. While the extent of Flvcr down-regulation was dose-responsive, a significant decrease (57%) occurred even with the lowest LPS dose (10 ng/ml). Macrophages decreased Fpn1 expression (71%) and increased Hmox1 expression (55%) in response to LPS stimulation as previously reported. Similar results were obtained with LPS from a different bacterial source (Salmonella minnesota Re595). We also performed these studies using primary macrophages cultured from murine bone marrow mononuclear cells and observed a similar decrease in Flvcr and Fpn1 (64 and 72%) and an increase in Hmox1 (40%) transcripts after stimulation with both O111:B4 and Re595 LPS.
While Fpn1 transcriptional regulation by heme and oxidative stress has been studied, the mechanism by which LPS regulates Fpn1 transcription is less clear. The similar pattern and kinetics of LPS-induced Flvcr and Fpn1 expression changes raise the possibility that the same regulatory mechanism is responsible. Analysis of the human and mouse Flvcr promoter regions revealed several putative LPS downstream transcription factor binding sites including NF-κB, AP1, and C/EBPβ. In addition to transcriptional regulation, LPS downstream signaling could alter Flvcr and Fpn1 mRNA stability and translation, so we compared the 5' untranslated regions (UTR) and 3'UTR of murine Flvcr and Fpn1. We found little similarity between the 5'UTR of Flvcr and the 5'UTR of Fpn1, known to contain an iron-responsive element (IRE) and be regulated by iron via iron regulatory proteins (IRP). However, alignment of the 3'UTR from Flvcr and Fpn1 showed similarity (pair wise score 65). Both the Flvcr and Fpn1 3'UTR are predicted to have a high degree of secondary structure based on their large negative fold energies (−421.25 and −300.74 kcal/mol), further suggesting that these 3'UTR may have a regulatory function. Studies are underway to determine the roles of the Flvcr promoter, 5'UTR, and 3'UTR in LPS-induced down-regulation.
This work suggests that LPS-induced down-regulation of Flvcr and Fpn1 might act in concert to decrease heme and iron export from macrophages and sequester iron from bacterial pathogens. Heme export control through FLVCR could serve as a novel mechanism of iron regulation in response to infection.
Disclosures:
No relevant conflicts of interest to declare.
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