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' 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' 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.

Disturbances of iron homeostasis are associated with altered susceptibility to infectious disease, but the underlying molecular mechanisms are poorly understood. To study this phenomenon, we examined innate immunity to oral Salmonella infection in Hfe knockout (Hfe(-/-)) mice, a model of the human inherited disorder of iron metabolism type I hemochromatosis. Salmonella- and LPS-induced inflammatory responses were attenuated in the mutant animals, with less severe enterocolitis observed in vivo and reduced macrophage TNF-alpha and IL-6 secretion measured in vitro. The macrophage iron exporter ferroportin (FPN) was up-regulated in the Hfe(-/-) mice, and correspondingly, intramacrophage iron levels were lowered. Consistent with the functional importance of these changes, the abnormal cytokine production of the mutant macrophages could be reproduced in wild-type cells by iron chelation, and in a macrophage cell line by overexpression of FPN. The results of analyzing specific steps in the biosynthesis of TNF-alpha and IL-6, including intracellular concentrations, posttranslational stability and transcript levels, were consistent with reduced translation of cytokine mRNAs in Hfe(-/-) macrophages. Polyribosome profile analysis confirmed that elevated macrophage FPN expression and low intracellular iron impaired the translation of specific inflammatory cytokine transcripts. Our results provide molecular insight into immune function in type I hemochromatosis and other disorders of iron homeostasis, and reveal a novel role for iron in the regulation of the inflammatory response.