Tumor Necrosis Factor α Inhibits Expression of the Iron Regulating Hormone Hepcidin in Murine Models of Innate Colitis

Background: Abnormal expression of the liver peptide hormone hepcidin, a key regulator of iron homeostasis, contributes to the pathogenesis of anemia in conditions such as inflammatory bowel disease (IBD). Since little is known about the mechanisms that control hepcidin expression during states of intestinal inflammation, we sought to shed light on this issue using mouse models. Methodology/Principal Findings: Hepcidin expression was evaluated in two types of intestinal inflammation caused by innate immune activation—dextran sulfate sodium (DSS)-induced colitis in wild-type mice and the spontaneous colitis occurring in T-bet/Rag2-deficient (TRUC) mice. The role of tumor necrosis factor (TNF) $\alpha$ was investigated by in vivo neutralization, and by treatment of a hepatocyte cell line, as well as mice, with the recombinant cytokine. Expression and activation of Smad1, a positive regulator of hepcidin transcription, were assessed during colitis and following administration or neutralization of TNF$\alpha$. Hepcidin expression progressively decreased with time during DSS colitis, correlating with changes in systemic iron distribution. TNF$\alpha$ inhibited hepcidin expression in cultured hepatocytes and non-colitic mice, while TNF$\alpha$ neutralization during DSS colitis increased it. Similar results were obtained in TRUC mice. These effects involved a TNF$\alpha$-dependent decrease in Smad1 protein but not mRNA. Conclusions/Significance: TNF$\alpha$ inhibits hepcidin expression in two distinct types of innate colitis, with down-regulation of Smad1 protein playing an important role in this process. This inhibitory effect of TNF$\alpha$ may be superseded by other factors in the context of T cell-mediated colitis given that in the latter form of intestinal inflammation hepcidin is usually up-regulated

Regulation of Lipopolysaccharide-Induced Translation of Tumor Necrosis Factor-Alpha by the Toll-Like Receptor 4 Adaptor Protein TRAM

Lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)-alpha requires the recruitment of two pairs of adaptors to the Toll-like receptor 4 cytoplasmic domain. The contribution of one pair - Toll-interleukin-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) and TRIF-related adaptor molecule (TRAM) - to TNF-alpha expression is not well understood. To clarify this issue, we studied TRAM knockout bone marrow-derived macrophages (BMDM). LPS-stimulated TRAM-deficient BMDM had decreased TNF-alpha protein expression even at times when TNF-alpha mRNA levels were normal, suggesting impaired translation. Consistent with this idea, knockdown of TRAM in RAW264.7 macrophages decreased translation of a reporter controlled by the TNF-alpha 3\u27 untranslated region, while transfection of TRAM in HEK293T cells increased translation of this reporter. Also consistent with a role for TRAM in TNF-alpha translation, LPS-induced activation of MK2, a kinase involved in this process, was impaired in TRAM-deficient BMDM. TRIF did not increase translation of the TNF-alpha 3\u27 untranslated region reporter when expressed in HEK293T cells. However, BMDM that lacked functional TRIF produced reduced levels of TNF-alpha protein in response to LPS despite normal amounts of the mRNA. Unlike BMDM, LPS-stimulated TRAM-deficient peritoneal macrophages displayed equivalent reductions in TNF-alpha protein and mRNA. Our results indicate that TRAM- and TRIF-dependent signals have a previously unappreciated, cell type-specific role in regulating TNF-alpha translation

Siderocalin inhibits the intracellular replication of Mycobacterium tuberculosis in macrophages

Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis (M.tb) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show that siderocalin expression is up-regulated following M.tb infection of mouse macrophage cell lines and primary murine alveolar macrophages. Furthermore, siderocalin added exogenously as a recombinant protein or over-expressed in the RAW264.7 macrophage cell line inhibited the intracellular growth of the pathogen. A variant form of siderocalin, which is expressed only in the macrophage cytosol, inhibited intracellular M.tb growth as effectively as the normal, secreted form, an observation that provides mechanistic insight into how siderocalin might influence iron acquisition by the bacteria in the phagosome. Our findings are consistent with an important role for siderocalin in protection against M.tb infection and suggest that exogenously administered siderocalin may have therapeutic applications in tuberculosis

Low dietary iron intake restrains the intestinal inflammatory response and pathology of enteric infection by food-borne bacterial pathogens

Orally administrated iron is suspected to increase susceptibility to enteric infections among children in infection endemic regions. Here we investigated the effect of dietary iron on the pathology and local immune responses in intestinal infection models. Mice were held on iron-deficient, normal iron, or high iron diets and after 2 weeks they were orally challenged with the pathogen Citrobacter rodentium. Microbiome analysis by pyrosequencing revealed profound iron- and infection-induced shifts in microbiota composition. Fecal levels of the innate defensive molecules and markers of inflammation lipocalin-2 and calprotectin were not influenced by dietary iron intervention alone, but were markedly lower in mice on the iron-deficient diet after infection. Next, mice on the iron-deficient diet tended to gain more weight and to have a lower grade of colon pathology. Furthermore, survival of the nematode Caenorhabditis elegans infected with Salmonella enterica serovar Typhimurium was prolonged after iron deprivation. Together, these data show that iron limitation restricts disease pathology upon bacterial infection. However, our data also showed decreased intestinal inflammatory responses of mice fed on high iron diets. Thus additionally, our study indicates that the effects of iron on processes at the intestinal host-pathogen interface may highly depend on host iron status, immune status, and gut microbiota composition.</p

Alterations of iron metabolism in DSS colitis.

<p><b>A,</b> Hepcidin mRNA levels, normalized to GAPDH, in livers of control, untreated mice (Con), or mice treated for 3 or 7 days with DSS. Expression is indicated relative to the mean of the control animals. *p = 0.01, n = 7 mice per group. <b>B,</b> Serum transaminase levels in control, untreated mice (Con), and mice treated for 7 days with DSS. n = 10 for the control group and 8 in the DSS treated group. <b>C,</b> Splenic iron concentrations in control, untreated mice (Con), or mice treated for 3 or 7 days with DSS. n = 7 in each group. <b>D,</b> Serum iron concentrations in control, untreated mice (Con), or mice treated for 3 or 7 days with DSS. n = 4 (control), 5 (3d DSS), 4 (7d DSS).</p

Effects of TNFα on hepcidin expression in Huh7 cells.

<p><b>A</b>, Huh7 cells were treated with the indicated concentrations of TNFα for 12 hours and hepcidin expression, normalized to actin, was determined relative to the mean of untreated cells. *p<0.0001, n = 5 for each condition. <b>B</b>, Huh7 cells were treated for 12 hours with 10 ng/ml of IL-6, either alone or together with 10 ng/ml of TNFα, and hepcidin expression, normalized to actin, was determined relative to the mean of control, untreated cells. *p = 0.014, n = 3 for each condition. <b>C</b>, Huh7 cells were transfected with a firefly luciferase reporter under the control of the hepcidin promoter along with a constitutively expressed Renilla reporter. Forty-eight hours after transfection, the cells were treated with 10 ng/ml of TNFα for 12 hours and luciferase activities determined. The ratio of firefly to Renilla (F/R) luciferase activity is indicated. *p = 0.002, n = 3 for each condition.</p