Heightened expression of TNFalpha by hepatic macrophages (HM) and peripheral blood monocytes (PBM) is a hallmark pathogenetic event in alcoholic hepatitis (AH). Although several hypotheses have been proposed, the precise molecular mechanism underlying the dysregulated TNFalpha expression in AH is still elusive. We have recently disclosed a novel signaling event involving a transient rise in the intracellular level of low molecular weight iron complex(es) ([LMW-Fe]i) for LPS-induced activation of IkB kinase and NF-kB, and TNFalpha expression in HM. We further demonstrated that [LMW-Fe]I signaling was accentuated, in parallel to increased non-heme iron content and heightened TNFalpha expression, in HM from rats with experimental alcoholic liver disease. The identical phenotype was also disclosed by us in the murine macrophage cell line (RAW.264.7) that is deficient in Nrampl, the protein that controls iron flux from endosomes, due to its G169D mutation. Finally, the treatment with a lipophilic iron chelator corrected both accentuated [LMW-Fe]i signaling and heightened TNFalpha expression in both examples. ? ? The present exploratory study will test the demonstrated novel link between [LMW-Fe]i signaling and ? TNFalpha expression, in PBM of AH patients to determine whether the [LMW-Fe]i signaling is primed as the molecular basis for the heightened TNFalpha response. The study will also examine in PBM of AH patients, the expression of Nrampl and other putative genes known to be involved in iron metabolism and homeostasis in order to search for the mechanisms of iron accumulation and consequent enhancement in [LMW-Fe]I signaling. To address these two specific aims, we will isolate PBM from patients with stable acute AH and gender and age-matched healthy subjects as the proven surrogate marker for HM. Iron content, [LMW-Fe]I signaling, and TNFalpha expression will be examined ex vivo. Real-time PCR and Western blot analysis will be performed on PBM to determine the expression of Nrampl, DMT1 (divalent metal transporter), ferroportin, TfR1 (transferrin receptor 1), HFE (a hemochromatosis gene product), ferritin H and L chains to correlate them with observed changes in iron content and [LMW-Fe]i signaling. Additional longitudinal comparison will be made between the acute AH phase and the remission phase in the same patients to determine whether they ? have inherent abnormalities in [LMW-Fe]i signaling or expression of iron regulatory molecules. ? ? In summary, the current proposal is the first exploratory study to test dysregulation of the novel [LMW-Fe]I signaling in AH patients as the molecular basis for heightened TNFalpha expression. ? ? ?
