Mice deficient in the hemochromatosis gene, Hfe, have attenuated inflammatory responses to Salmonella infection associated with decreased macrophage TNF-α and IL-6 biosynthesis after exposure to LPS. In this study, we show that the abnormal cytokine production is related to impaired TLR4 signaling. Despite their abnormal response to LPS, Hfe KO macrophages produced amounts of TNF-α similar to those in WT cells after TLR2 stimulation. Consistent with this finding, LPS-induced activation of Mal/MyD88-dependent events was normal in the mutant macrophages. However, LPS-induced IFN-β expression, a TRAM/TRIF-dependent response activated by TLR4, was reduced by Hfe deficiency. This reduction could be replicated in WT macrophages with the use of iron chelators. In contrast, TLR3-activated expression of IFN-β, a TRIF-dependent response, was normal in Hfe KO macrophages and was unaffected by iron chelation. Our data suggest that low intracellular iron selectively impairs signaling via the TLR4/TRAM/TRIF pathway proximal to TRIF and results in reduced LPS-induced cytokine expression. Furthermore, by mimicking the altered iron metabolism associated with Hfe deficiency, we found that 3 different inhibitors of hepcidin attenuated Salmonella-induced and noninfectious enterocolitis. Thus, manipulation of iron homeostasis could represent a new therapeutic approach to controlling inflammation.
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-α 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-α 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.
Necrotizing enterocolitis is a devastating inflammatory condition of the intestine that occurs almost exclusively in premature newborns. Although its exact pathogenesis is unclear, we have postulated that it may result from a predisposition of the immature intestine to mount an unusually robust and damaging response to microbial infection. In support of this idea, we report that the IL-8 response of an immature human enterocyte cell line to bacterial infection was significantly higher than that of a mature enterocyte cell line. The response in both cell lines was flagellin-dependent. Corresponding to the difference in IL-8 production, the immature enterocytes expressed appreciably lower levels of specific IB genes when compared with the mature enterocytes. Similar developmentally regulated differences in cytokine response and IB expression were also seen in primary rat enterocytes, indicating that these observations were not peculiarities of the cell lines. Furthermore, when the level of IB␣ expression was increased in the immature cell line by transfection, the flagellin-dependent IL-8 response was attenuated. Thus, we have demonstrated a previously undescribed developmental regulation of IB expression in the intestine involved in modulating the IL-8 response to bacterial infection, which may contribute to the pathogenesis of age-specific inflammatory bowel diseases such as necrotizing enterocolitis. E nterocytes are active participants in host defense against microbial invasion. Certain aspects of this function are developmentally regulated, resulting in distinct differences between the immature and mature intestine with respect to interactions with microorganisms (1-4). Immaturity of epithelial barrier function, relative deficiencies in the expression of antimicrobial factors, and developmental variations in the pattern of epithelial surface glycosylation all contribute to the susceptibility of early postnatal intestine to bacterial infection (5-7). Concurrently, the secretion of proinflammatory cytokines is greater at earlier stages of development than in the adult. Specifically, IL-8 production in response to IL-1 and tumor necrosis factor ␣ has been shown to be significantly increased in the human fetal intestinal epithelial cell (IEC) line H4 and fetal intestinal organ cultures compared with the adult IEC line Caco2 and biopsies from older children (8). The combination of weaker antimicrobial defense mechanisms and exaggerated cytokine responses may render the immature gut prone to extensive inflammatory damage after infection. These properties could thus contribute to the pathogenesis of age-specific diseases such as necrotizing enterocolitis (NEC), an ischemic and inflammatory bowel necrosis that primarily affects premature neonates after the initiation of enteral feeding.We have postulated a model of NEC pathogenesis in which early feeding results in exposure to numbers and types of bacteria that the premature intestine is ill-equipped to handle (9). The exaggerated inflammatory responses that are cha...
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