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Liver Iron Loading in Alcohol-Associated Liver Disease

Open AccessPublished:October 24, 2022DOI:https://doi.org/10.1016/j.ajpath.2022.08.010
      Alcohol-associated liver disease (ALD) is a common chronic liver disease with increasing incidence worldwide. Alcoholic liver steatosis/steatohepatitis can progress to liver fibrosis/cirrhosis, which can cause predisposition to hepatocellular carcinoma. Also, ALD diagnosis and management are confounded by several challenges. Iron loading is a feature of ALD. It can exacerbate alcohol-induced liver injury and promote ALD pathologic progression. Knowledge of the mechanisms that mediate liver iron loading can help identify cellular/molecular targets and thereby aid in designing adjunct diagnostic, prognostic, and therapeutic approaches for ALD. Therefore, herein, we reviewed the cellular mechanisms underlying alcohol-induced liver iron loading and discussed how excess iron in patients with ALD can promote liver fibrosis and aggravate disease pathology. Essentially, alcohol-induced increase in hepatic transferrin receptor-1 expression and up-regulation of high iron protein in Kupffer cells (proposed) facilitate iron deposition and retention in the liver. Iron is loaded in both parenchymal and nonparenchymal liver cells. Iron-loaded liver can promote ferroptosis and thereby contribute to ALD pathology. Iron and alcohol can independently elevate oxidative stress. Therefore, a combination of excess iron and alcohol amplifies oxidative stress and accelerates liver injury. Also, via secretion of proinflammatory and profibrotic factors, excess iron–stimulated hepatocytes directly or indirectly (through Kupffer cell activation) activate the hepatic stellate cells. Persistently activated hepatic stellate cells promote liver fibrosis, and thereby facilitate ALD progression.
      Alcohol consumption is increasing worldwide, and so is the incidence of alcohol-associated liver disease (ALD).
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      Global alcohol exposure between 1990 and 2017 and forecasts until 2030: a modelling study.
      With no standard laboratory diagnostic test to confirm ALD etiology, asymptomatic early stages, and high costs of disease management, ALD continues to pose challenges on all fronts. Abstinence is the only curative option.
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      • Mehta K.J.
      Betaine in ameliorating alcohol-induced hepatic steatosis.
      Iron loading is one of the characteristic features of ALD. Even mild to moderate alcohol consumption increases liver iron content.
      • Harrison-Findik D.D.
      Role of alcohol in the regulation of iron metabolism.
      This can aggravate alcohol-induced liver injury via various mechanisms and promote the pathologic progression of the disease. Knowledge of these mechanisms that mediate liver iron increment in ALD and its consequences at cellular level may help identify cellular/molecular targets and thereby aid in designing better diagnostic, prognostic, and therapeutic approaches for ALD. Such investigations have proved useful in the past. For example, a study showed that liver iron content exhibited a negative correlation with the survival of patients with ALD, and was thus predictive of mortality in patients with alcoholic cirrhosis.
      • Ganne-Carrié N.
      • Christidis C.
      • Chastang C.
      • Ziol M.
      • Chapel F.
      • Imbert-Bismut F.
      • Trinchet J.C.
      • Guettier C.
      • Beaugrand M.
      Liver iron is predictive of death in alcoholic cirrhosis: a multivariate study of 229 consecutive patients with alcoholic and/or hepatitis C virus cirrhosis: a prospective follow up study.
      Therefore, herein, we reviewed the cellular mechanisms underlying alcohol-induced liver iron loading and discussed how excess iron in patients with ALD can promote liver fibrosis and aggravate disease pathology.

      High Liver Iron Content in ALD

      Patients with ALD/chronic alcohol consumers often show high hepatic iron levels.
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      • Taylor S.L.
      • Reyes V.
      • Raaka S.
      • Berger J.
      • Kowdley K.V.
      Hepatic iron overload in alcoholic end-stage liver disease is associated with iron deposition in other organs in the absence of HFE-1 hemochromatosis.
      • Kohgo Y.
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      • Ikuta K.
      • Suzuki Y.
      • Hosoki Y.
      • Saito H.
      • Kato J.
      Iron accumulation in alcoholic liver diseases.
      • Kowdley K.V.
      Iron overload in patients with chronic liver disease.
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      • Halliday J.W.
      • Powell L.W.
      Association between alcoholism and increased hepatic iron stores.
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      • Hoffbrand A.V.
      • Sherlock S.
      Hepatic iron stores and markers of iron overload in alcoholics and patients with idiopathic hemochromatosis.
      About 50% of patients with ALD tend to show liver iron excess.
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      • Rausch V.
      The role of iron in alcohol-mediated hepatocarcinogenesis.
      A study showed that the mean liver iron content (measured as μg/100 mg dry weight) in alcoholics was 156.4 ± 7.8, which was significantly higher than in the controls (53 ± 7).
      • Chapman R.W.
      • Morgan M.Y.
      • Laulicht M.
      • Hoffbrand A.V.
      • Sherlock S.
      Hepatic iron stores and markers of iron overload in alcoholics and patients with idiopathic hemochromatosis.
      Alcoholic cirrhotic patients frequently show high liver iron content, which is associated with increased mortality.
      • Ganne-Carrié N.
      • Christidis C.
      • Chastang C.
      • Ziol M.
      • Chapel F.
      • Imbert-Bismut F.
      • Trinchet J.C.
      • Guettier C.
      • Beaugrand M.
      Liver iron is predictive of death in alcoholic cirrhosis: a multivariate study of 229 consecutive patients with alcoholic and/or hepatitis C virus cirrhosis: a prospective follow up study.
      Increment in liver iron occurs not only because of alcohol consumption but also because of additional factors and mechanisms involving the second hit, such as a high-fat diet in combination with alcohol consumption. Regardless, high liver iron content can contribute to permanent liver injury and hepatocellular carcinoma.
      • Varghese J.
      • James J.V.
      • Sagi S.
      • Chakraborty S.
      • Sukumaran A.
      • Ramakrishna B.
      • Jacob M.
      Decreased hepatic iron in response to alcohol may contribute to alcohol-induced suppression of hepcidin.
      Indeed, with increased serum iron in alcohol consumers, there could be iron deposition in extrahepatic organs, such as the pancreas and heart, as seen in other iron-loaded conditions.
      • Nam H.
      • Wang C.-Y.
      • Zhang L.
      • Zhang W.
      • Hojyo S.
      • Fukada T.
      • Knutson M.D.
      ZIP14 and DMT1 in the liver, pancreas, and heart are differentially regulated by iron deficiency and overload: implications for tissue iron uptake in iron-related disorders.
      For example, an autopsy of a 54-year–old woman with ALD showed iron overload in the liver as well as the pancreas, heart, and stomach.
      • Eng S.C.
      • Taylor S.L.
      • Reyes V.
      • Raaka S.
      • Berger J.
      • Kowdley K.V.
      Hepatic iron overload in alcoholic end-stage liver disease is associated with iron deposition in other organs in the absence of HFE-1 hemochromatosis.

      Pattern of Iron Deposition in Hepatic Cells in ALD

      There are two different proposals with regard to iron deposition in the different cell types of the liver. According to one proposal, in mild ALD, iron is preferably deposited in the hepatocytes (parenchymal cells of the liver). But as the condition progresses to severe ALD, iron loading is more obvious in the Kupffer cells (nonparenchymal cells in liver) compared with hepatocytes.
      • Kohgo Y.
      • Ohtake T.
      • Ikuta K.
      • Suzuki Y.
      • Hosoki Y.
      • Saito H.
      • Kato J.
      Iron accumulation in alcoholic liver diseases.
      Pietrangelo
      • Pietrangelo A.
      Iron-induced oxidant stress in alcoholic liver fibrogenesis.
      has also supported the idea of nonparenchymal iron loading in the advanced stages of alcoholic liver fibrogenesis. In contrast, another proposal suggests that in secondary iron overload syndromes, such as ALD, iron accumulates in the reticuloendothelial system, which includes the Kupffer cells of the liver, and accumulates in the hepatocytes after the reticuloendothelial cells are saturated with iron.
      • İdilman İ.S.
      • Akata D.
      • Özmen M.N.
      • Karçaaltıncaba M.
      Different forms of iron accumulation in the liver on MRI.
      Regardless, in ALD, iron deposition is observed in both hepatocytes and Kupffer cells (ie, in parenchymal and nonparenchymal cells of the liver).

      Cellular Mechanisms that Increase Liver Iron in ALD

      Hepcidin, the liver-secreted iron hormone, is the regulator of systemic iron homeostasis.
      • Sangkhae V.
      • Nemeth E.
      Regulation of the iron homeostatic hormone hepcidin.
      Alcohol-induced suppression of hepcidin expression is the main cause of systemic iron loading in alcohol consumers. Serum iron loading is further supported by alcohol-induced elevations in the expressions of iron transporters in the duodenum: duodenal divalent metal transporter 1 (DMT1) and ferroportin. These events enhance intestinal iron absorption (ie, increase iron entry into the circulation),
      • Bridle K.
      • Cheung T.-K.
      • Murphy T.
      • Walters M.
      • Anderson G.
      • Crawford D.G.
      • Fletcher L.M.
      Hepcidin is down-regulated in alcoholic liver injury: implications for the pathogenesis of alcoholic liver disease.
      • Costa-Matos L.
      • Batista P.
      • Monteiro N.
      • Simões M.
      • Egas C.
      • Pereira J.
      • Pinho H.
      • Santos N.
      • Ribeiro J.
      • Cipriano M.A.
      • Henriques P.
      • Girão F.
      • Rodrigues A.
      • Carvalho A.
      Liver hepcidin mRNA expression is inappropriately low in alcoholic patients compared with healthy controls.
      • Dostalikova-Cimburova M.
      • Balusikova K.
      • Kratka K.
      • Chmelikova J.
      • Hejda V.
      • Hnanicek J.
      • Neubauerova J.
      • Vranova J.
      • Kovar J.
      • Horak J.
      Role of duodenal iron transporters and hepcidin in patients with alcoholic liver disease.
      • Harrison-Findik D.D.
      Is the iron regulatory hormone hepcidin a risk factor for alcoholic liver disease?.
      and this forms the basis for liver iron loading in alcohol consumers.
      There are several mechanisms/cellular events that facilitate liver iron loading in ALD. These have been depicted in Figure 1.
      Figure thumbnail gr1
      Figure 1Cellular events underlying alcohol-induced iron loading in different cell types. Alcohol consumption decreases hepcidin levels in the circulation. In turn, this increases intestinal absorption of iron. Elevated serum iron levels cause iron deposition in various cell types, including the hepatocytes and Kupffer cells in the liver, via elevation in transferrin receptor-1 (TfR1) and high iron (HFE; proposed). Also, alcohol-induced elevation of non–transferrin-bound iron (NTBI) transporters ZIP and DMT1 on hepatocytes, as observed in some studies, aids in hepatocyte iron loading. Green arrows with yellow stars indicate variability in results with regard to alcohol-induced elevation of these NTBI transporters.

      Increased Hepatic TfR1

      One mechanism involves increment in hepatic transferrin receptor-1 (TfR1). Cellular TfR1 is the receptor for circulating iron-bound transferrin. It facilitates the entry of transferrin-bound iron (TBI) into various cells. In a study, most habitual alcohol consumers/patients with ALD showed increased expression of hepatic TfR1 (in hepatocytes), unlike healthy liver tissues.
      • Suzuki Y.
      • Saito H.
      • Suzuki M.
      • Hosoki Y.
      • Sakurai S.
      • Fujimoto Y.
      • Kohgo Y.
      Up-regulation of transferrin receptor expression in hepatocytes by habitual alcohol drinking is implicated in hepatic iron overload in alcoholic liver disease.
      Alcohol-induced oxidative stress increases the activity of iron regulatory proteins (IRPs), and this is thought to be partly responsible for this increase in TfR1 expression.
      • Kohgo Y.
      • Ohtake T.
      • Ikuta K.
      • Suzuki Y.
      • Hosoki Y.
      • Saito H.
      • Kato J.
      Iron accumulation in alcoholic liver diseases.
      ,
      • Kohgo Y.
      • Ohtake T.
      • Ikuta K.
      • Suzuki Y.
      • Torimoto Y.
      • Kato J.
      Dysregulation of systemic iron metabolism in alcoholic liver diseases.
      Kupffer cells of alcohol-fed rodents have also shown about sixfold and ninefold increases in TfR1 gene and protein expressions, respectively.
      • Xiong S.
      • She H.
      • Zhang A.-S.
      • Wang J.
      • Mkrtchyan H.
      • Dynnyk A.
      • Gordeuk V.R.
      • French S.W.
      • Enns C.A.
      • Tsukamoto H.
      Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis.
      This collectively indicates that alcohol-induced elevation in TfR1 expression promotes iron uptake in both parenchymal and nonparenchymal cells of the liver (Figure 1). Thus, TfR1 up-regulation may partly explain the liver iron loading in patients with ALD.
      • Suzuki Y.
      • Saito H.
      • Suzuki M.
      • Hosoki Y.
      • Sakurai S.
      • Fujimoto Y.
      • Kohgo Y.
      Up-regulation of transferrin receptor expression in hepatocytes by habitual alcohol drinking is implicated in hepatic iron overload in alcoholic liver disease.
      • Kohgo Y.
      • Ohtake T.
      • Ikuta K.
      • Suzuki Y.
      • Torimoto Y.
      • Kato J.
      Dysregulation of systemic iron metabolism in alcoholic liver diseases.
      • Xiong S.
      • She H.
      • Zhang A.-S.
      • Wang J.
      • Mkrtchyan H.
      • Dynnyk A.
      • Gordeuk V.R.
      • French S.W.
      • Enns C.A.
      • Tsukamoto H.
      Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis.
      Interestingly, alcohol alone treatment to VL-17A cells neither altered the expressions of TfR1 and IRP2 nor altered IRP1 RNA binding activity.
      • Harrison-Findik D.D.
      • Schafer D.
      • Klein E.
      • Timchenko N.A.
      • Kulaksiz H.
      • Clemens D.
      • Fein E.
      • Andriopoulos B.
      • Pantopoulos K.
      • Gollan J.
      Alcohol metabolism-mediated oxidative stress down-regulates hepcidin transcription and leads to increased duodenal iron transporter expression.
      However, a combination of alcohol and iron treatment to rat primary hepatocytes increased the expression of TfR1 (compared with iron alone treatment) partly through the increased activity of IRPs.
      • Harrison-Findik D.D.
      • Schafer D.
      • Klein E.
      • Timchenko N.A.
      • Kulaksiz H.
      • Clemens D.
      • Fein E.
      • Andriopoulos B.
      • Pantopoulos K.
      • Gollan J.
      Alcohol metabolism-mediated oxidative stress down-regulates hepcidin transcription and leads to increased duodenal iron transporter expression.
      ,
      • Suzuki M.
      • Fujimoto Y.
      • Suzuki Y.
      • Hosoki Y.
      • Saito H.
      • Nakayama K.
      • Ohtake T.
      • Kohgo Y.
      Induction of transferrin receptor by ethanol in rat primary hepatocyte culture.
      On the basis of this, it can be extrapolated that the increased TfR1 expression observed in alcohol consumers is a result of combined effect of alcohol and iron.
      Normally, the intracellularly operating IRP–iron response element system regulates cellular iron levels by acting on the transcripts for various iron-related genes, including TfR1. Under cellular iron excess, the IRP–iron response element system functions to reduce cellular TfR1 to reduce TBI entry into the cells.
      • Muckenthaler M.U.
      • Galy B.
      • Hentze M.W.
      Systemic iron homeostasis and the iron-responsive element/iron-regulatory protein (IRE/IRP) regulatory network.
      Alcohol-induced increment in hepatic TfR1 expression in the presence of hepatic iron loading suggests that alcohol can disturb the aforementioned TfR1-regulatory mechanism and cause or contribute to increased hepatocellular iron uptake.
      • Kohgo Y.
      • Ohtake T.
      • Ikuta K.
      • Suzuki Y.
      • Hosoki Y.
      • Saito H.
      • Kato J.
      Iron accumulation in alcoholic liver diseases.
      ,
      • Kohgo Y.
      • Ohtake T.
      • Ikuta K.
      • Suzuki Y.
      • Torimoto Y.
      • Kato J.
      Dysregulation of systemic iron metabolism in alcoholic liver diseases.
      Macrophages also show iron loading. These cells predominantly acquire iron due to phagocytosis of senescent red blood cells. However, these cells do express DMT1, TfR1, hemoglobin scavenger receptor (CD163), and natural resistance-associated macrophage protein 1. These proteins are involved in iron uptake and transport,
      • Harrison-Findik D.D.
      Is the iron regulatory hormone hepcidin a risk factor for alcoholic liver disease?.
      and may contribute to the increment in liver iron levels.

      Putative Role of HFE Protein

      The high iron (HFE) protein may contribute to liver iron accumulation. HFE is a cell surface protein that appears to exhibit multiple functions. First, the HFE can bind to TfR2 to form an iron-sensing complex on the cell membrane. This complex regulates/induces hepcidin expression.
      • Gao J.
      • Chen J.
      • Kramer M.
      • Tsukamoto H.
      • Zhang A.-S.
      • Enns C.A.
      Interaction of the hereditary hemochromatosis protein HFE with transferrin receptor 2 is required for transferrin-induced hepcidin expression.
      Hereby, HFE functions as a regulator of hepcidin transcription. Second, HFE can affect the binding of iron-bound transferrin to TfR1. When HFE binds to TfR1, the affinity of TfR1 to bind to iron-bound transferrin is reduced,
      • Feder J.N.
      • Penny D.M.
      • Irrinki A.
      • Lee V.K.
      • Lebrón J.A.
      • Watson N.
      • Tsuchihashi Z.
      • Sigal E.
      • Bjorkman P.J.
      • Schatzman R.C.
      The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding.
      and this reduces cellular iron uptake. Hereby, HFE functions as a regulator of cellular iron uptake. Third, HFE has shown to inhibit cellular iron efflux. Stable transfection-expression of HFE in human colonic carcinoma cell line increased cellular ferritin expression, indicating intracellular iron accumulation/elevation. However, this was not due to transferrin-dependent iron uptake. This suggested that the HFE expression prevented cellular iron efflux and facilitated intracellular iron retention, which resulted in the aforementioned intracellular ferritin elevation.
      • Davies P.S.
      • Enns C.A.
      Expression of the hereditary hemochromatosis protein HFE increases ferritin levels by inhibiting iron export in HT29 cells.
      Ferroportin is the sole known iron transporter (exporter) on the surfaces of various cell types, including the hepatocytes and Kupffer cells. It has been postulated that HFE can interact with ferroportin and inhibit cellular iron release from macrophages (Figure 1).
      • Drakesmith H.
      • Sweetland E.
      • Schimanski L.
      • Edwards J.
      • Cowley D.
      • Ashraf M.
      • Bastin J.
      • Townsend A.R.M.
      The hemochromatosis protein HFE inhibits iron export from macrophages.
      Alcohol activates HFE gene transcription in the Kupffer cells.
      • Xiong S.
      • She H.
      • Zhang A.-S.
      • Wang J.
      • Mkrtchyan H.
      • Dynnyk A.
      • Gordeuk V.R.
      • French S.W.
      • Enns C.A.
      • Tsukamoto H.
      Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis.
      Alcohol-exposed rat Kupffer cells showed increased Hfe mRNA levels.
      • Harrison-Findik D.D.
      Is the iron regulatory hormone hepcidin a risk factor for alcoholic liver disease?.
      On the basis of the postulated function of HFE, this may reduce/inhibit cellular iron export and facilitate iron retention within the Kupffer cells. This may be an additional mechanism causing liver iron loading under the influence of alcohol (Figure 1). Interestingly, duodenal HFE mRNA expression in patients with ALD with iron overload (defined as increased ferritin or transferrin saturation) was significantly higher than in controls, unlike the expression levels in patients with ALD without iron overload and patients with ALD with anemia, in whom levels were similar to controls.
      • Dostalikova-Cimburova M.
      • Balusikova K.
      • Kratka K.
      • Chmelikova J.
      • Hejda V.
      • Hnanicek J.
      • Neubauerova J.
      • Vranova J.
      • Kovar J.
      • Horak J.
      Role of duodenal iron transporters and hepcidin in patients with alcoholic liver disease.
      This suggested that increment in duodenal HFE expression was linked with systemic iron loading, and this could subsequently lead to iron deposition in the liver and other organs. On the basis of these data, it appears that HFE function may be cell specific: mediating intracellular iron retention in one cell type, as postulated in case of Kupffer cells, while allowing systemic iron loading through duodenal cells. This hypothesis on the cell-specific nature of HFE needs to be confirmed.

      Enigma Around Ethanol-Induced NTBI Uptake

      Depending on the form of iron [TBI or non–transferrin-bound iron (NTBI)], cellular iron uptake can occur via two main mechanisms: TBI uptake and NTBI uptake. NTBI uptake occurs independent of TfR1 and contributes to cell toxicity when in excess. It involves NTBI transporters, such as DMT1, zinc transporter ZIP14 (on hepatocytes), and ZIP8, and some L-type calcium channels in the cardiomyocytes that are believed to be involved in NTBI uptake.
      • Brissot P.
      • Pietrangelo A.
      • Adams P.C.
      • de Graaff B.
      • McLaren C.E.
      • Loréal O.
      Haemochromatosis.
      TBI uptake is regulated by the IRP–iron response element system
      • Muckenthaler M.U.
      • Galy B.
      • Hentze M.W.
      Systemic iron homeostasis and the iron-responsive element/iron-regulatory protein (IRE/IRP) regulatory network.
      and functions by down-regulating TfR1 expression under excess iron conditions. In contrast, NTBI uptake occurs despite iron loading. Hepatocytes and parenchymal cells of other tissues, like pancreas and heart, are prone to NTBI uptake. This explains iron loading in the liver and other organs.
      • Brissot P.
      • Pietrangelo A.
      • Adams P.C.
      • de Graaff B.
      • McLaren C.E.
      • Loréal O.
      Haemochromatosis.
      ZIP14 and DMT1 can mediate NTBI uptake in hepatocytes (Figure 1). In the context of the effect of alcohol on these NTBI transporters and NTBI uptake, there have been some apparently differing observations. For example, in mice, chronic alcohol and/or iron feeding (15 weeks) caused significantly elevated levels of NTBI in serum and increased the expressions of hepatic DMT1 and ZIP14 at both mRNA and protein levels. This explained the observed increment in their liver iron content
      • Tang Y.
      • Li Y.
      • Yu H.
      • Gao C.
      • Liu L.
      • Xing M.
      • Liu L.
      • Yao P.
      Quercetin attenuates chronic ethanol hepatotoxicity: implication of “free” iron uptake and release.
      and indicated alcohol-induced elevation in NTBI and in NTBI uptake. Furthermore, in human HepaRG cells (hepatic cell line), ethanol increased total iron content, and this appeared to be mediated via elevations in the gene expression of DMT1 and TfR1,
      • Do T.H.T.
      • Gaboriau F.
      • Cannie I.
      • Batusanski F.
      • Ropert M.
      • Moirand R.
      • Brissot P.
      • Loreal O.
      • Lescoat G.
      Iron-mediated effect of alcohol on hepatocyte differentiation in HepaRG cells.
      indicating the utility of both NTBI and TBI uptake in the presence of alcohol.
      However, in other studies, ethanol exposure dramatically reduced hepatic ZIP14 protein levels in mice,
      • Sun Q.
      • Li Q.
      • Zhong W.
      • Zhang J.
      • Sun X.
      • Tan X.
      • Yin X.
      • Sun X.
      • Zhang X.
      • Zhou Z.
      Dysregulation of hepatic zinc transporters in a mouse model of alcoholic liver disease.
      and there was no major change in hepatic DMT1 in mice after 12 weeks of alcohol feeding.
      • Varghese J.
      • James J.V.
      • Sagi S.
      • Chakraborty S.
      • Sukumaran A.
      • Ramakrishna B.
      • Jacob M.
      Decreased hepatic iron in response to alcohol may contribute to alcohol-induced suppression of hepcidin.
      Because the data are variable, it would be interesting to further investigate and clarify the significance and role of NTBI uptake under the influence of alcohol.

      Alcohol and Liver Ferritin: Some Contradictions

      Ferritin (the iron storage protein present intracellularly and in the circulation) is elevated in response to elevation in iron and/or inflammation. It is composed of two types of chains: heavy (H) and light (L). Rats fed with alcohol for 7 weeks showed significantly increased levels of H-ferritin expression in the liver.
      • Harrison-Findik D.D.
      • Klein E.
      • Crist C.
      • Evans J.
      • Timchenko N.
      • Gollan J.
      Iron-mediated regulation of liver hepcidin expression in rats and mice is abolished by alcohol.
      Similarly, alcohol treatment to HepG2 cells increased the expressions of both H and L ferritin and alcohol increased L-ferritin synthesis in rat hepatocytes.
      • Moirand R.
      • Kerdavid F.
      • Loréal O.
      • Hubert N.
      • Leroyer P.
      • Brissot P.
      • Lescoat G.
      Regulation of ferritin expression by alcohol in a human hepatoblastoma cell line and in rat hepatocyte cultures.
      Also, alcohol exposure to human hepatoma HepaRG cell line increased the expression of L-ferritin.
      • Tuoi Do T.H.
      • Gaboriau F.
      • Ropert M.
      • Moirand R.
      • Cannie I.
      • Brissot P.
      • Loréal O.
      • Lescoat G.
      Ethanol effect on cell proliferation in the human hepatoma HepaRG cell line: relationship with iron metabolism.
      Such an alcohol-induced increase in liver ferritin could be either a rescue mechanism to combat the alcohol-induced elevation in iron levels and store excess iron or it could be a response to alcohol-induced inflammation or both.
      However, a study in mice fed with alcohol for 12 weeks showed decreased hepatic L-ferritin expression, and there were no significant effects at the earlier time points.
      • Varghese J.
      • James J.V.
      • Sagi S.
      • Chakraborty S.
      • Sukumaran A.
      • Ramakrishna B.
      • Jacob M.
      Decreased hepatic iron in response to alcohol may contribute to alcohol-induced suppression of hepcidin.
      Likewise, in VL-17A cells, alcohol did not alter the expression of H-ferritin.
      • Harrison-Findik D.D.
      • Schafer D.
      • Klein E.
      • Timchenko N.A.
      • Kulaksiz H.
      • Clemens D.
      • Fein E.
      • Andriopoulos B.
      • Pantopoulos K.
      • Gollan J.
      Alcohol metabolism-mediated oxidative stress down-regulates hepcidin transcription and leads to increased duodenal iron transporter expression.
      These differential ferritin responses to alcohol require further investigation.

      Combination of Excess Iron and Alcohol Enhances Oxidative Stress and Aggravates ALD Pathology

      Under physiological conditions, normal levels of reactive oxygen species (ROS) produced by cellular mechanisms are utilized for cellular purposes, and excess ROS are scavenged/tackled by the endogenous antioxidant mechanisms to prevent ROS-mediated injury. However, excess free iron can accelerate the Fenton reaction, leading to the production of large amounts of ROS that saturate the endogenous antioxidant mechanisms. These free radicals increase oxidative stress and can cause immense cellular and tissue damage
      • Mehta K.
      Chapter 4 - oxidative stress in iron toxicity of the liver.
      by acting on cellular organelles, DNA, proteins, and lipids.
      Both iron overload and alcohol can independently cause oxidative stress and lipid peroxidation. Thus, excess free iron and alcohol act in a synergistic manner to cause liver damage, and the combined effect exacerbates liver injury.
      • Harrison-Findik D.D.
      Is the iron regulatory hormone hepcidin a risk factor for alcoholic liver disease?.
      The fibrogenic potential of iron is enhanced when it acts with other hepatotoxins, like alcohol. The catalytic free iron could directly add to the hepatoxicity of alcohol and/or amplify the generation of cytokines and fibrogenic medFigiators from the nearby Kupffer cells. This means that slight increase in tissue iron levels in the presence of alcohol (and other metabolites) can accelerate fibrogenesis and advance the liver disease. In the early stages of liver disease, iron-loaded hepatocytes release profibrogenic cytokines and sustain fibrogenesis, whereas at the advanced stages, fibrogenesis is primarily governed by iron-induced hepatocellular necrosis.
      • Pietrangelo A.
      Iron-induced oxidant stress in alcoholic liver fibrogenesis.
      Thus, in ALD, excess iron can enhance liver injury by acting as a cofactor for liver fibrogenesis. Also, the combined oxidative stress caused by alcohol and excess iron may cause DNA damage and mutations, resulting in increased predisposition to liver cancer.

      Ferroptosis in Context

      Ferroptosis: An Iron-Dependent Cell Death

      Ferroptosis is a form of regulated cell death that is morphologically and biochemically distinct from other cell death patterns, like apoptosis, autophagy, and pyroptosis. Its normal physiological function has not been established yet, but it has a role in pathology. Distinct from its role in hepatocellular carcinoma, where it increases sensitivity to sorafenib (used for liver cancer treatment), in chronic liver diseases, including ALD, ferroptosis aggravates hepatic damage. Generally, it has been implicated in the pathology of liver diseases via several signaling pathways.
      • Stockwell B.R.
      • Friedmann Angeli J.P.
      • Bayir H.
      • Bush A.I.
      • Conrad M.
      • Dixon S.J.
      • Fulda S.
      • Gascón S.
      • Hatzios S.K.
      • Kagan V.E.
      • Noel K.
      • Jiang X.
      • Linkermann A.
      • Murphy M.E.
      • Overholtzer M.
      • Oyagi A.
      • Pagnussat G.C.
      • Park J.
      • Ran Q.
      • Rosenfeld C.S.
      • Salnikow K.
      • Tang D.
      • Torti F.M.
      • Torti S.V.
      • Toyokuni S.
      • Woerpel K.A.
      • Zhang D.D.
      Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease.
      ,
      • Wu J.
      • Wang Y.
      • Jiang R.
      • Xue R.
      • Yin X.
      • Wu M.
      • Meng Q.
      Ferroptosis in liver disease: new insights into disease mechanisms.
      Ferroptosis is iron-dependent cell death and is characterized by excessive iron accumulation and lipid peroxidation.
      • Wu J.
      • Wang Y.
      • Jiang R.
      • Xue R.
      • Yin X.
      • Wu M.
      • Meng Q.
      Ferroptosis in liver disease: new insights into disease mechanisms.
      Essentially, during ferroptosis, there is ample oxidation-reduction–active iron present, and glutathione peroxidase is unable to efficiently execute its antioxidant action and repair lipid peroxidation, resulting in unrestricted lipid peroxidation and iron-dependent accumulation of high levels of lipid hydroperoxides.
      • Stockwell B.R.
      • Friedmann Angeli J.P.
      • Bayir H.
      • Bush A.I.
      • Conrad M.
      • Dixon S.J.
      • Fulda S.
      • Gascón S.
      • Hatzios S.K.
      • Kagan V.E.
      • Noel K.
      • Jiang X.
      • Linkermann A.
      • Murphy M.E.
      • Overholtzer M.
      • Oyagi A.
      • Pagnussat G.C.
      • Park J.
      • Ran Q.
      • Rosenfeld C.S.
      • Salnikow K.
      • Tang D.
      • Torti F.M.
      • Torti S.V.
      • Toyokuni S.
      • Woerpel K.A.
      • Zhang D.D.
      Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease.
      ,
      • Capelletti M.M.
      • Manceau H.
      • Puy H.
      • Peoc’h K.
      Ferroptosis in liver diseases: an overview.

      Role of Ferroptosis in ALD Pathology

      Alcohol metabolism generates a large amount of acetaldehyde, reduces the levels of the antioxidant glutathione in the mitochondria, and increases ROS production, followed by elevated lipid peroxidation in liver cells. Studies confirm that alcohol treatment induces excessive accumulation of iron in the liver, and increases ROS accompanied by lipid peroxidation, thereby initiating ferroptosis.
      • Liu C.-Y.
      • Wang M.
      • Yu H.-M.
      • Han F.-X.
      • Wu Q.-S.
      • Cai X.-J.
      • Kurihara H.
      • Chen Y.-X.
      • Li Y.-F.
      • He R.-R.
      Ferroptosis is involved in alcohol-induced cell death in vivo and in vitro.
      ,
      • Zhou Z.
      • Ye T.J.
      • DeCaro E.
      • Buehler B.
      • Stahl Z.
      • Bonavita G.
      • Daniels M.
      • You M.
      Intestinal SIRT1 deficiency protects mice from ethanol-induced liver injury by mitigating ferroptosis.
      The key features of ferroptosis are iron and lipid peroxidation. Both liver iron loading and lipid disorder are features of ALD,
      • Wu J.
      • Wang Y.
      • Jiang R.
      • Xue R.
      • Yin X.
      • Wu M.
      • Meng Q.
      Ferroptosis in liver disease: new insights into disease mechanisms.
      which generates a strong reason for ferroptosis initiation in the livers of patients with ALD.
      As previously discussed, excess iron generates free radicals and enhances oxidative stress/injury, and the liver is prone to oxidative injury in general. Thus, ferroptosis has a pathogenic role in excess iron–induced hepatic damage and fibrosis, and excess iron is a risk factor for liver fibrosis and cirrhosis.
      • Chen J.
      • Li X.
      • Ge C.
      • Min J.
      • Wang F.
      The multifaceted role of ferroptosis in liver disease.
      This explains the role of iron overload in inducing ferroptosis and thereby contributing to ALD pathology.

      Effect of Ferroptosis on Hepatocytes

      Long-term alcohol consumption can cause liver iron loading and subsequently promote ferroptosis in the hepatocytes. Hepatocytes have myriads of functions, including regulation of systemic levels of iron, glucose, and lipoproteins. So, regardless of the form of cell death (ferroptosis or otherwise), hepatocyte death or dysfunction is a critical factor for liver injury and failure. Hepatocytes that undergo ferroptosis burst and release damage-associated molecular patterns. These are proinflammatory in nature and activate NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes in the Kupffer cells, leading to the release of a large volume of proinflammatory cytokines
      • Li L.-X.
      • Guo F.-F.
      • Liu H.
      • Zeng T.
      Iron overload in alcoholic liver disease: underlying mechanisms, detrimental effects, and potential therapeutic targets.
      that aggravate disease pathology.
      Thus, excess iron, as found in ALD livers, can induce oxidative stress, cause iron-dependent cell death ferroptosis, promote inflammation, and thereby contribute to liver injury. Unsurprisingly, iron as an initiator of ferroptosis is linked with mortality related to ALD.
      • Liu C.-Y.
      • Wang M.
      • Yu H.-M.
      • Han F.-X.
      • Wu Q.-S.
      • Cai X.-J.
      • Kurihara H.
      • Chen Y.-X.
      • Li Y.-F.
      • He R.-R.
      Ferroptosis is involved in alcohol-induced cell death in vivo and in vitro.
      Ferroptosis inhibitors, like ferrostatin-1, can rescue the alcohol-induced hepatocyte death and limit alcohol-induced liver injury.
      • Chen S.
      • Zhu J.
      • Zang X.
      • Zhai Y.
      The emerging role of ferroptosis in liver diseases.
      Therefore, ferroptosis appears to be a promising target for ameliorating ALD pathology.

      Cell-Specific Effect of Ferroptosis

      Unlike the aforementioned situation, where ferroptosis in hepatocytes exerts a pathologic effect and inhibition of ferroptosis in the hepatocytes is therapeutic, ferroptosis in hepatic stellate cells (HSCs) shows a completely opposite effect. Several studies in animal models have shown that ferroptosis in activated HSCs can reduce liver fibrosis and exert a curative effect. Also, blocking ferroptosis in the HSCs can promote liver fibrosis. Thus, the effect of ferroptosis appears to be cell type specific. This presents challenges at the therapeutic front because selectively targeting ferroptosis in HSCs can be difficult.
      • Zhou X.
      • Fu Y.
      • Liu W.
      • Mu Y.
      • Zhang H.
      • Chen J.
      • Liu P.
      Ferroptosis in chronic liver diseases: opportunities and challenges.
      To enable this, specialized systems that exclusively target the HSCs will be required.

      Links between Alcohol, Autophagy, Ferritinophagy, and Ferroptosis

      Autophagy: A Cell Survival Mechanism that Can also Promote Cell Death

      Autophagy is a conserved catabolic cellular process that is triggered following an insult or stress. It degrades damaged organelles and extra/unnecessary proteins, aiming to maintain a balance between protein degradation, synthesis, and recycling of cellular components. The process involves the formation of vesicles called autophagosomes, which deliver the cytosolic cargo to lysosomes for degradation, and then the material is recycled back to the cytosol. Dysregulation of autophagy has been implicated in metabolic and neurodegenerative diseases, inflammation, aging, and cancer. In the liver, autophagy maintains the cellular functionality of hepatocytes.
      • Condello M.
      • Pellegrini E.
      • Caraglia M.
      • Meschini S.
      Targeting autophagy to overcome human diseases.
      ,
      • Dolganiuc A.
      • Thomes P.G.
      • Ding W.-X.
      • Lemasters J.J.
      • Donohue T.M.
      Autophagy in alcohol-induced liver diseases.

      Autophagy Degrades Ferritin

      Autophagy degrades ferritin, the iron-storage protein. This is called ferritinophagy. Ferritin degradation inside the autolysosomes leads to release of iron from ferritin. This released free iron is likely to be transported back to cytosol, leading to increment in ROS and oxidative stress, and this can trigger ferroptosis. Thus, ferritinophagy can play a role in triggering ferroptosis (Figure 2),
      • Liu J.
      • Guo Z.-N.
      • Yan X.-L.
      • Huang S.
      • Ren J.-X.
      • Luo Y.
      • Yang Y.
      Crosstalk between autophagy and ferroptosis and its putative role in ischemic stroke.
      • Zhou Y.
      • Shen Y.
      • Chen C.
      • Sui X.
      • Yang J.
      • Wang L.
      • Zhou J.
      The crosstalk between autophagy and ferroptosis: what can we learn to target drug resistance in cancer?.
      • Ajoolabady A.
      • Aslkhodapasandhokmabad H.
      • Libby P.
      • Tuomilehto J.
      • Lip G.Y.H.
      • Penninger J.M.
      • Richardson D.R.
      • Tang D.
      • Zhou H.
      • Wang S.
      • Klionsky D.J.
      • Kroemer G.
      • Ren J.
      Ferritinophagy and ferroptosis in the management of metabolic diseases.
      • Park E.
      • Chung S.W.
      ROS-mediated autophagy increases intracellular iron levels and ferroptosis by ferritin and transferrin receptor regulation.
      • Tang M.
      • Chen Z.
      • Wu D.
      • Chen L.
      Ferritinophagy/ferroptosis: iron-related newcomers in human diseases.
      and ferritin negatively regulates ferroptosis.
      • Kang R.
      • Tang D.
      Autophagy and ferroptosis - what's the connection?.
      In HepG2 cells, autophagy inhibition increased ferritin heavy chain production.
      • Zhao Y.
      • Lu J.
      • Mao A.
      • Zhang R.
      • Guan S.
      Autophagy inhibition plays a protective role in ferroptosis induced by alcohol via the p62-Keap1-Nrf2 pathway.
      In theory, this would aid in scavenging/accommodating free iron within ferritin, leading to reduction in oxidative stress and, thereby, reduction in ferroptosis. Collectively, data suggest that ferritinophagy can promote ALD pathology, in part via ferroptosis, because ferroptosis aggravates liver pathology (Figure 2).
      Figure thumbnail gr2
      Figure 2Putative associations between autophagy, ferritinophagy, and ferroptosis under the influence of alcohol. Alcohol seems to have a dual effect of autophagy (ie, it can both stimulate and impair autophagy). This differential effect of alcohol on autophagy and the consequent ambiguity is indicated through an asterisk in the figure. Degradation of ferritin via autophagy is ferritinophagy. Ferritinophagy can trigger ferroptosis, whereas increment in ferritin can increase the probability of accommodating free iron, thereby reducing excess iron–induced oxidative stress, and consequently reducing ferroptosis. Autophagy can also trigger ferroptosis through ferritinophagy-independent routes, such as those involving frataxin deficiency, and degradation of damaged or excess cellular components that eventually increases free iron levels and/or lipid peroxidation.
      • Liu J.
      • Guo Z.-N.
      • Yan X.-L.
      • Huang S.
      • Ren J.-X.
      • Luo Y.
      • Yang Y.
      Crosstalk between autophagy and ferroptosis and its putative role in ischemic stroke.
      ,
      • Zhou Y.
      • Shen Y.
      • Chen C.
      • Sui X.
      • Yang J.
      • Wang L.
      • Zhou J.
      The crosstalk between autophagy and ferroptosis: what can we learn to target drug resistance in cancer?.
      Interestingly, although autophagy can trigger ferroptosis, which can exacerbate alcohol-associated liver disease (ALD) pathology, autophagy appears to also impart a protective effect and decrease or blunt ALD pathology. These apparently opposing concepts have been indicated by question marks in the figure and need further clarification.

      Autophagy Shows Divergent Relation with ALD: Further Clarity Needed

      Apparently, there are differing data in the context of the effect of alcohol of autophagy. Studies indicate that alcohol exposure can increase autophagosome formation and trigger autophagy. This is a protective mechanism that selectively removes damaged mitochondria and hepatic lipids. However, alcohol can also impair lysosome function or lysosomal biogenesis, leading to deficient autophagy in the hepatocytes, and contribute to ALD pathology (Figure 2).
      • Chao X.
      • Ding W.-X.
      Role and mechanisms of autophagy in alcohol-induced liver injury.
      These apparently contrasting effects could be due to differential effects of acute and chronic alcohol on autophagy, due to differential effects of alcohol itself on autophagy, or involve the fact that autophagy can mediate both cell survival and cell death; the latter may depend on cell type and cell context.
      • Das G.
      • Shravage B.V.
      • Baehrecke E.H.
      Regulation and function of autophagy during cell survival and cell death.
      The reason/s for these differential effects need to be identified.
      In addition, there can be apparently conflicting inferences involving autophagy, ferroptosis, and ALD pathology (Figure 2). Inhibition of autophagy in alcohol-fed mice increased hepatoxicity, steatosis, oxidative stress, and hepatocyte apoptosis, and activation of autophagy blunted the alcohol-induced steatosis.
      • Chao X.
      • Ding W.-X.
      Role and mechanisms of autophagy in alcohol-induced liver injury.
      This presents a protective role of autophagy under alcoholic conditions. However, experiments in HepG2 cells showed that inhibition/impairment of autophagy activates the p62-Keap1-Nrf2 pathway. This is protective against alcohol-induced ferroptosis,
      • Zhao Y.
      • Lu J.
      • Mao A.
      • Zhang R.
      • Guan S.
      Autophagy inhibition plays a protective role in ferroptosis induced by alcohol via the p62-Keap1-Nrf2 pathway.
      and thereby should reduce/decelerate ALD pathology. Unlike the previous scenario, this presents autophagy impairment as having a protective role under alcoholic conditions (Figure 2).
      These conflicting relationships, which infer that autophagy can trigger ferroptosis but also decrease ALD pathology, and impaired autophagy can reduce ferroptosis but also accelerate ALD pathology, which requires clarification.

      Intercellular Events Underlying Iron-Aggravated Liver Fibrosis in ALD

      Iron loading is one of the independent risk factors for fibrosis in ALD.
      • Raynard B.
      • Balian A.
      • Fallik D.
      • Capron F.
      • Bedossa P.
      • Chaput J.-C.
      • Naveau S.
      Risk factors of fibrosis in alcohol-induced liver disease.
      Thus, it is important to review the intercellular events involved in the iron-facilitated progression to liver fibrosis.
      Figure 3 summarizes the intercellular interactions, and the ways in which iron loading can exacerbate liver injury in ALD and promote liver fibrosis. Table 1
      • Mehta K.J.
      • Farnaud S.J.
      • Sharp P.A.
      Iron and liver fibrosis: mechanistic and clinical aspects.
      • Miyata T.
      • Nagy L.E.
      Programmed cell death in alcohol-associated liver disease.
      • Yang Y.M.
      • Cho Y.E.
      • Hwang S.
      Crosstalk between oxidative stress and inflammatory liver injury in the pathogenesis of alcoholic liver disease.
      • Gerjevic L.N.
      • Liu N.
      • Lu S.
      • Harrison-Findik D.D.
      Alcohol activates TGF-beta but inhibits BMP receptor-mediated Smad signaling and Smad4 binding to hepcidin promoter in the liver.
      • Seo W.
      • Jeong W.-I.
      Hepatic non-parenchymal cells: master regulators of alcoholic liver disease?.
      • Kanamori Y.
      • Tanaka M.
      • Itoh M.
      • Ochi K.
      • Ito A.
      • Hidaka I.
      • Sakaida I.
      • Ogawa Y.
      • Suganami T.
      Iron-rich Kupffer cells exhibit phenotypic changes during the development of liver fibrosis in NASH.
      • Xiong S.
      • She H.
      • Sung C.K.
      • Tsukamoto H.
      Iron-dependent activation of NF-kappaB in Kupffer cells: a priming mechanism for alcoholic liver disease.
      • Bloomer S.A.
      • Brown K.E.
      Iron-induced liver injury: a critical reappraisal.
      • Hoeft K.
      • Bloch D.B.
      • Graw J.A.
      • Malhotra R.
      • Ichinose F.
      • Bagchi A.
      Iron loading exaggerates the inflammatory response to the toll-like receptor 4 ligand lipopolysaccharide by altering mitochondrial homeostasis.
      • Mehta K.J.
      • Coombes J.D.
      • Briones-Orta M.
      • Manka P.P.
      • Williams R.
      • Patel V.B.
      • Syn W.-K.
      Iron enhances hepatic fibrogenesis and activates transforming growth factor-[beta] signaling in murine hepatic stellate cells.
      • Philippe M.A.
      • Ruddell R.G.
      • Ramm G.A.
      Role of iron in hepatic fibrosis: one piece in the puzzle.
      • Ruddell R.G.
      • Hoang-Le D.
      • Barwood J.M.
      • Rutherford P.S.
      • Piva T.J.
      • Watters D.J.
      • Santambrogio P.
      • Arosio P.
      • Ramm G.A.
      Ferritin functions as a proinflammatory cytokine via iron-independent protein kinase C zeta/nuclear factor kappaB–regulated signaling in rat hepatic stellate cells.
      • Petrillo S.
      • Manco M.
      • Altruda F.
      • Fagoonee S.
      • Tolosano E.
      Liver sinusoidal endothelial cells at the crossroad of iron overload and liver fibrosis.
      • Parrow N.L.
      • Fleming R.E.
      Liver sinusoidal endothelial cells as iron sensors.
      • Addo L.
      • Tanaka H.
      • Yamamoto M.
      • Toki Y.
      • Ito S.
      • Ikuta K.
      • Sasaki K.
      • Ohtake T.
      • Torimoto Y.
      • Fujiya M.
      • Kohgo Y.
      Hepatic nerve growth factor induced by iron overload triggers defenestration in liver sinusoidal endothelial cells.
      • Lim P.J.
      • Duarte T.L.
      • Arezes J.
      • Garcia-Santos D.
      • Hamdi A.
      • Pasricha S.-R.
      • Armitage A.E.
      • Mehta H.
      • Wideman S.
      • Santos A.G.
      • Santos-Gonçalves A.
      • Morovat A.
      • Hughes J.R.
      • Soilleux E.
      • Wang C.-Y.
      • Bayer A.L.
      • Klenerman P.
      • Willberg C.B.
      • Hartley R.C.
      • Murphy M.P.
      • Babitt J.L.
      • Ponka P.
      • Porto G.
      • Drakesmith H.
      Nrf2 controls iron homoeostasis in haemochromatosis and thalassaemia via Bmp6 and hepcidin.
      • Lafoz E.
      • Ruart M.
      • Anton A.
      • Oncins A.
      • Hernández-Gea V.
      The endothelium as a driver of liver fibrosis and regeneration.
      presents an overview of the effect of iron overload on some of the core cell types in the liver. Each cell type of the hepatic lobule is actively involved in the fibrogenic process. The main cell types involved in this process are the hepatocytes, Kupffer cells, and HSCs, whereas the liver endothelial cells (Table 1) and fat-storing cells (explained in the subsequent section) also play a role.
      Figure thumbnail gr3
      Figure 3Intercellular events depicting the role of iron in enhancing alcohol-induced liver fibrosis. Alcohol can cause iron loading in the hepatocytes and Kupffer cells. Oxidative injury to hepatocytes due to excess iron and alcohol can lead to hepatocyte death. Kupffer cells phagocytose dead/damaged hepatocytes and get activated. Activated Kupffer cells release profibrotic cytokines and activate the hepatic stellate cells (HSCs). In addition, profibrotic/inflammatory cytokines released from injured hepatocytes together with reactive oxygen species (ROS) and acetaldehyde produced from alcohol metabolism in the hepatocytes activate the HSCs. Following activation, HSCs secrete profibrotic factors and excessive extracellular matrix that collectively form the basis for liver fibrosis. Adipocytes also play a role in promoting alcohol-induced liver fibrosis, and together with excess iron, the pathology may be aggravated. β-FGF, β-fibroblast growth factor; IFN-γ, interferon-γ; MCP-1, monocyte chemoattractant protein-1; PDGF, platelet-derived growth factor; α-SMA, α-smooth muscle actin; TGF, transforming growth factor; TNF-α, tumor necrosis factor-α.
      Table 1Overview of the Most Prominent Effects of Iron Overload on the Core Liver Cells and the Associated Underlying Mechanisms
      Liver cell type and its generic functionProminent effects of iron overloadUnderlying cellular mechanisms in context of iron loading
      Hepatocytes (hepatic parenchymal cells, make majority of liver parenchyma and exhibit various functions, including sensing iron in the circulation and secreting the iron-regulating hormone hepcidin)
      • Sangkhae V.
      • Nemeth E.
      Regulation of the iron homeostatic hormone hepcidin.
      Increased oxidative stress, resulting in damage to cellular organelles, lipids, proteins, and DNA.
      • Li L.-X.
      • Guo F.-F.
      • Liu H.
      • Zeng T.
      Iron overload in alcoholic liver disease: underlying mechanisms, detrimental effects, and potential therapeutic targets.
      ,
      • Mehta K.J.
      • Farnaud S.J.
      • Sharp P.A.
      Iron and liver fibrosis: mechanistic and clinical aspects.
      Excess iron–induced elevation in ROS production is via the Fenton reaction.
      • Li L.-X.
      • Guo F.-F.
      • Liu H.
      • Zeng T.
      Iron overload in alcoholic liver disease: underlying mechanisms, detrimental effects, and potential therapeutic targets.
      ,
      • Mehta K.J.
      • Farnaud S.J.
      • Sharp P.A.
      Iron and liver fibrosis: mechanistic and clinical aspects.
      Cell deathExcess ROS causes lipid peroxidation, which contributes to different types of cell deaths, including ferroptosis,
      • Mehta K.J.
      • Farnaud S.J.
      • Sharp P.A.
      Iron and liver fibrosis: mechanistic and clinical aspects.
      • Miyata T.
      • Nagy L.E.
      Programmed cell death in alcohol-associated liver disease.
      • Yang Y.M.
      • Cho Y.E.
      • Hwang S.
      Crosstalk between oxidative stress and inflammatory liver injury in the pathogenesis of alcoholic liver disease.
      Increased synthesis and secretion of hepcidin
      • Sangkhae V.
      • Nemeth E.
      Regulation of the iron homeostatic hormone hepcidin.
      Hepcidin is induced via the BMP-SMAD pathway.
      • Sangkhae V.
      • Nemeth E.
      Regulation of the iron homeostatic hormone hepcidin.
      (Notably in ALD, hepcidin synthesis and secretion is reduced due to alcohol-induced inhibition of the BMP-SMAD pathway,
      • Gerjevic L.N.
      • Liu N.
      • Lu S.
      • Harrison-Findik D.D.
      Alcohol activates TGF-beta but inhibits BMP receptor-mediated Smad signaling and Smad4 binding to hepcidin promoter in the liver.
      attenuation of JAK/STAT signaling,
      • Bridle K.
      • Cheung T.-K.
      • Murphy T.
      • Walters M.
      • Anderson G.
      • Crawford D.G.
      • Fletcher L.M.
      Hepcidin is down-regulated in alcoholic liver injury: implications for the pathogenesis of alcoholic liver disease.
      and oxidative stress.
      • Harrison-Findik D.D.
      Is the iron regulatory hormone hepcidin a risk factor for alcoholic liver disease?.
      ,
      • Li L.-X.
      • Guo F.-F.
      • Liu H.
      • Zeng T.
      Iron overload in alcoholic liver disease: underlying mechanisms, detrimental effects, and potential therapeutic targets.
      )
      Kupffer cells (hepatic nonparenchymal cells, clear microorganisms, dead cells, debris, and circulating endotoxin)
      • Seo W.
      • Jeong W.-I.
      Hepatic non-parenchymal cells: master regulators of alcoholic liver disease?.
      Increased production of inflammatory cytokines
      • Kanamori Y.
      • Tanaka M.
      • Itoh M.
      • Ochi K.
      • Ito A.
      • Hidaka I.
      • Sakaida I.
      • Ogawa Y.
      • Suganami T.
      Iron-rich Kupffer cells exhibit phenotypic changes during the development of liver fibrosis in NASH.
      Iron loading can activate NF-κB,
      • Li L.-X.
      • Guo F.-F.
      • Liu H.
      • Zeng T.
      Iron overload in alcoholic liver disease: underlying mechanisms, detrimental effects, and potential therapeutic targets.
      ,
      • Xiong S.
      • She H.
      • Sung C.K.
      • Tsukamoto H.
      Iron-dependent activation of NF-kappaB in Kupffer cells: a priming mechanism for alcoholic liver disease.
      which can stimulate the production of proinflammatory cytokines, like TNF-α and IL-6.
      • Bloomer S.A.
      • Brown K.E.
      Iron-induced liver injury: a critical reappraisal.
      Enhanced inflammatory response to LPS
      • Xiong S.
      • She H.
      • Zhang A.-S.
      • Wang J.
      • Mkrtchyan H.
      • Dynnyk A.
      • Gordeuk V.R.
      • French S.W.
      • Enns C.A.
      • Tsukamoto H.
      Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis.
      ,
      • Hoeft K.
      • Bloch D.B.
      • Graw J.A.
      • Malhotra R.
      • Ichinose F.
      • Bagchi A.
      Iron loading exaggerates the inflammatory response to the toll-like receptor 4 ligand lipopolysaccharide by altering mitochondrial homeostasis.
      Disruption of mitochondrial homeostasis and increased generation of mitochondrial superoxide partly promote inflammatory response to LPS.
      • Hoeft K.
      • Bloch D.B.
      • Graw J.A.
      • Malhotra R.
      • Ichinose F.
      • Bagchi A.
      Iron loading exaggerates the inflammatory response to the toll-like receptor 4 ligand lipopolysaccharide by altering mitochondrial homeostasis.
      HSCs (hepatic nonparenchymal cells, generally quiescent, mediate wound healing following an injury)Persistent cell activation and proliferation, leading to promotion of fibrosis
      • Mehta K.J.
      • Farnaud S.J.
      • Sharp P.A.
      Iron and liver fibrosis: mechanistic and clinical aspects.
      Stimulation of the expressions of type I collagen and α-SMA (makers of fibrosis), increased production of TGF-β1, and activation of TGF-β pathway.
      • Mehta K.J.
      • Coombes J.D.
      • Briones-Orta M.
      • Manka P.P.
      • Williams R.
      • Patel V.B.
      • Syn W.-K.
      Iron enhances hepatic fibrogenesis and activates transforming growth factor-[beta] signaling in murine hepatic stellate cells.
      ,
      • Philippe M.A.
      • Ruddell R.G.
      • Ramm G.A.
      Role of iron in hepatic fibrosis: one piece in the puzzle.
      Extracellular ferritin stimulates inflammatory pathway in HSCs.
      • Ruddell R.G.
      • Hoang-Le D.
      • Barwood J.M.
      • Rutherford P.S.
      • Piva T.J.
      • Watters D.J.
      • Santambrogio P.
      • Arosio P.
      • Ramm G.A.
      Ferritin functions as a proinflammatory cytokine via iron-independent protein kinase C zeta/nuclear factor kappaB–regulated signaling in rat hepatic stellate cells.
      Activated HSCs exhibit a receptor for H-ferritin. Binding of ferritin (H-ferritin) activates NF-κB through PI3 kinase, PKCζ, MEK1/2, MAPK, and IKKα/β. Thereby, extracellular ferritin acts as a proinflammatory mediator.
      • Ruddell R.G.
      • Hoang-Le D.
      • Barwood J.M.
      • Rutherford P.S.
      • Piva T.J.
      • Watters D.J.
      • Santambrogio P.
      • Arosio P.
      • Ramm G.A.
      Ferritin functions as a proinflammatory cytokine via iron-independent protein kinase C zeta/nuclear factor kappaB–regulated signaling in rat hepatic stellate cells.
      LSECs (hepatic nonparenchymal cells, form a fenestrated endothelium that allows movement of selective molecules, and play a role in role in clearance of macromolecules from blood,
      • Seo W.
      • Jeong W.-I.
      Hepatic non-parenchymal cells: master regulators of alcoholic liver disease?.
      differentiated LSECs maintain HSC quiescence and help prevent fibrosis
      • Petrillo S.
      • Manco M.
      • Altruda F.
      • Fagoonee S.
      • Tolosano E.
      Liver sinusoidal endothelial cells at the crossroad of iron overload and liver fibrosis.
      )
      Induce hepcidin production in the hepatocytes
      • Parrow N.L.
      • Fleming R.E.
      Liver sinusoidal endothelial cells as iron sensors.
      LSECs can sense iron and produce BMPs in response. BMPs 2 and 6 can induce hepcidin synthesis in hepatocytes via BMP-SMAD pathway.
      • Sangkhae V.
      • Nemeth E.
      Regulation of the iron homeostatic hormone hepcidin.
      ,
      • Parrow N.L.
      • Fleming R.E.
      Liver sinusoidal endothelial cells as iron sensors.
      (Note that in ALD, hepcidin synthesis and secretion is reduced due to the previously explained reasons.)
      Following chronic liver injury (including persistent iron overload), LSECs can dedifferentiate and activate the HSCs, which leads to increased production of extracellular matrix, LSECs lose their fenestrations (defenestration) and function
      • Seo W.
      • Jeong W.-I.
      Hepatic non-parenchymal cells: master regulators of alcoholic liver disease?.
      ,
      • Petrillo S.
      • Manco M.
      • Altruda F.
      • Fagoonee S.
      • Tolosano E.
      Liver sinusoidal endothelial cells at the crossroad of iron overload and liver fibrosis.
      ,
      • Addo L.
      • Tanaka H.
      • Yamamoto M.
      • Toki Y.
      • Ito S.
      • Ikuta K.
      • Sasaki K.
      • Ohtake T.
      • Torimoto Y.
      • Fujiya M.
      • Kohgo Y.
      Hepatic nerve growth factor induced by iron overload triggers defenestration in liver sinusoidal endothelial cells.
      The effect of iron on LSEC defenestration is not direct. Iron-stimulated hepatocytes secrete nerve growth factor. This acts on nerve growth factor receptor on LSECs and triggers defenestration (in part).
      • Addo L.
      • Tanaka H.
      • Yamamoto M.
      • Toki Y.
      • Ito S.
      • Ikuta K.
      • Sasaki K.
      • Ohtake T.
      • Torimoto Y.
      • Fujiya M.
      • Kohgo Y.
      Hepatic nerve growth factor induced by iron overload triggers defenestration in liver sinusoidal endothelial cells.
      Also, excess iron–induced mitochondrial oxidative damage activates transcription factor Nrf2 in LSECs.
      • Lim P.J.
      • Duarte T.L.
      • Arezes J.
      • Garcia-Santos D.
      • Hamdi A.
      • Pasricha S.-R.
      • Armitage A.E.
      • Mehta H.
      • Wideman S.
      • Santos A.G.
      • Santos-Gonçalves A.
      • Morovat A.
      • Hughes J.R.
      • Soilleux E.
      • Wang C.-Y.
      • Bayer A.L.
      • Klenerman P.
      • Willberg C.B.
      • Hartley R.C.
      • Murphy M.P.
      • Babitt J.L.
      • Ponka P.
      • Porto G.
      • Drakesmith H.
      Nrf2 controls iron homoeostasis in haemochromatosis and thalassaemia via Bmp6 and hepcidin.
      Continuous activation of Nrf2 inhibits autophagy.
      • Petrillo S.
      • Manco M.
      • Altruda F.
      • Fagoonee S.
      • Tolosano E.
      Liver sinusoidal endothelial cells at the crossroad of iron overload and liver fibrosis.
      Normally, autophagy helps maintain LSEC phenotype (ie, fenestrae by controlling nitric oxide bioavailability).
      • Lafoz E.
      • Ruart M.
      • Anton A.
      • Oncins A.
      • Hernández-Gea V.
      The endothelium as a driver of liver fibrosis and regeneration.
      Thus, iron overload can cause LSEC defenestration over time.
      • Petrillo S.
      • Manco M.
      • Altruda F.
      • Fagoonee S.
      • Tolosano E.
      Liver sinusoidal endothelial cells at the crossroad of iron overload and liver fibrosis.
      ALD, alcohol-associated liver disease; HSC, hepatic stellate cell; LPS, lipopolysaccharide; LSEC, liver sinusoidal endothelial cell; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; ROS, reactive oxygen species; α-SMA, α-smooth muscle actin; TGF, transforming growth factor; TNF-α, tumor necrosis factor-α.

      Interaction between Hepatic Stellate Cells, Hepatocytes, and Kupffer Cells

      The HSCs play a crucial role in liver fibrogenesis. Activation of HSCs is a normal phenomenon that mediates wound repair. Following repair, HSCs either revert to their quiescent state or undergo apoptosis. However, persistent liver insults keep the HSCs continuously activated. These HSCs secrete excessive amounts of profibrogenic factors and extracellular matrix that collectively induce a pathologic state and form the basis of liver fibrosis. When liver iron exceeds 60 μmol/g, the HSCs get activated. Iron-induced promotion of fibrogenic mechanisms has been shown in murine HSCs, and the contribution of excess iron in enhancing liver fibrosis is well established.
      • Mehta K.J.
      • Farnaud S.J.
      • Sharp P.A.
      Iron and liver fibrosis: mechanistic and clinical aspects.
      ,
      • Mehta K.J.
      • Coombes J.D.
      • Briones-Orta M.
      • Manka P.P.
      • Williams R.
      • Patel V.B.
      • Syn W.-K.
      Iron enhances hepatic fibrogenesis and activates transforming growth factor-[beta] signaling in murine hepatic stellate cells.
      ,
      • Mehta K.
      • Farnaud S.
      • Patel V.B.
      Chapter 28 - molecular effects of alcohol on iron metabolism.
      Iron-loaded hepatocytes release profibrogenic factors and can directly activate the HSCs (Figure 3). In addition, these hepatocytes can release profibrotic/proinflammatory factors and stimulate the Kupffer cells.
      • Trinder D.
      • Fox C.
      • Vautier G.
      • Olynyk J.K.
      Molecular pathogenesis of iron overload.
      Alcohol increases the translocation of lipopolysaccharide from the intestine to the liver, which additionally stimulates the Kupffer cells. Once activated, the Kupffer cells release proinflammatory and profibrotic factors, such as tumor necrosis factor (TNF)-α, IL-1, IL-6, IL-8, IL-10, interferon-γ, transforming growth factor-β1, platelet-derived growth factor, β-fibroblast growth factor, monocyte chemoattractant protein-1, and ROS. These cytokines, in turn, activate the HSCs (Figure 3).
      • Trinder D.
      • Fox C.
      • Vautier G.
      • Olynyk J.K.
      Molecular pathogenesis of iron overload.
      • Sikorska K.
      • Bernat A.
      • Wróblewska A.
      Molecular pathogenesis and clinical consequences of iron overload in liver cirrhosis.
      • Slevin E.
      • Baiocchi L.
      • Wu N.
      • Ekser B.
      • Sato K.
      • Lin E.
      • Ceci L.
      • Chen L.
      • Lorenzo S.R.
      • Xu W.
      • Kyritsi K.
      • Meadows V.
      • Zhou T.
      • Kundu D.
      • Han Y.
      • Kennedy L.
      • Glaser S.
      • Francis H.
      • Alpini G.
      • Meng F.
      Kupffer cells: inflammation pathways and cell-cell interactions in alcohol-associated liver disease.
      • Zeng T.
      • Zhang C.-L.
      • Xiao M.
      • Yang R.
      • Xie K.-Q.
      Critical roles of Kupffer cells in the pathogenesis of alcoholic liver disease: from basic science to clinical trials.
      Essentially, injured hepatocytes can directly activate the HSCs or indirectly activate HSCs by stimulating the Kupffer cells to secrete profibrotic factors that, in turn, activate HSCs. Regardless, on activation, HSCs differentiate into myofibroblasts and synthesize and release excessive amounts of extracellular matrix composed of elastin, collagen, and other matrix proteins, thereby exhibiting liver fibrosis (Figure 3).
      • Mehta K.J.
      • Farnaud S.J.
      • Sharp P.A.
      Iron and liver fibrosis: mechanistic and clinical aspects.
      ,
      • Trinder D.
      • Fox C.
      • Vautier G.
      • Olynyk J.K.
      Molecular pathogenesis of iron overload.
      Activation of NF-κB correlates with liver inflammation and fibrosis in ALD.
      • Ribeiro P.S.
      • Cortez-Pinto H.
      • Solá S.
      • Castro R.E.
      • Ramalho R.M.
      • Baptista A.
      • Moura M.C.
      • Camilo M.E.
      • Rodrigues C.M.P.
      Hepatocyte apoptosis, expression of death receptors, and activation of NF-kappaB in the liver of nonalcoholic and alcoholic steatohepatitis patients.
      Alcohol-induced accumulation of iron in Kupffer cells can activate NF-κB and worsen experimental ALD/alcoholic steatohepatitis.
      • Xiong S.
      • She H.
      • Zhang A.-S.
      • Wang J.
      • Mkrtchyan H.
      • Dynnyk A.
      • Gordeuk V.R.
      • French S.W.
      • Enns C.A.
      • Tsukamoto H.
      Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis.
      ,
      • Xiong S.
      • She H.
      • Sung C.K.
      • Tsukamoto H.
      Iron-dependent activation of NF-kappaB in Kupffer cells: a priming mechanism for alcoholic liver disease.
      ,
      • Tsukamoto H.
      • Lin M.
      • Ohata M.
      • Giulivi C.
      • French S.W.
      • Brittenham G.
      Iron primes hepatic macrophages for NF-kappaB activation in alcoholic liver injury.
      Alcoholics show increased levels of lipopolysaccharide in the circulation. Iron and lipopolysaccharide are believed to activate NF-κB in the Kupffer cells and induce the synthesis of proinflammatory cytokines, like TNF-α.
      • Harrison-Findik D.D.
      Is the iron regulatory hormone hepcidin a risk factor for alcoholic liver disease?.
      TNF-α plays an important role in liver injury. Normally, hepatocytes are not negatively affected by TNF-α. However, alcohol sensitizes the hepatocytes to injury by TNF-α and causes hepatocyte cell death via apoptosis.
      • Zeng T.
      • Zhang C.-L.
      • Xiao M.
      • Yang R.
      • Xie K.-Q.
      Critical roles of Kupffer cells in the pathogenesis of alcoholic liver disease: from basic science to clinical trials.
      ,
      • Osna N.A.
      • Donohue T.M.
      • Kharbanda K.K.
      Alcoholic liver disease: pathogenesis and current management.
      These dead cells would be engulfed by the Kupffer cells (Figure 3). In animal models, on Kupffer cell depletion or inactivation, alcohol-induced effects, such as inflammation, fatty liver, and necrosis, were dampened. Thus, Kupffer cells play an important role in the pathologic progression of ALD.
      • Harrison-Findik D.D.
      Is the iron regulatory hormone hepcidin a risk factor for alcoholic liver disease?.

      The Adipocyte Context

      In addition to Kupffer cells and HSCs, other cells in the surrounding, such as the adipocytes from adipose tissue, are involved in ALD pathogenesis. Independent of the effect of alcohol, it has been speculated that lipid peroxidation by-products released from iron-overloaded hepatocytes are able to stimulate collagen gene transcription in the neighboring fat-storing cells directly or via activation of Kupffer cells.
      • Gualdi R.
      • Casalgrandi G.
      • Montosi G.
      • Ventura E.
      • Pietrangelo A.
      Excess iron into hepatocytes is required for activation of collagen type I gene during experimental siderosis.
      This may further aggravate ALD pathogenesis in cases with iron overload. Notably, excess iron–generated ROS and lipid peroxidation by-products can activate both Kupffer cells and HSCs (Figure 3).
      • Pietrangelo A.
      Iron-induced oxidant stress in alcoholic liver fibrogenesis.
      Also, alcohol induces inflammation in the adipose tissue. Alcohol-induced lipolysis in the adipocytes (which promotes hepatic steatosis) together with inflammatory responses in the macrophages release increased levels of free fatty acids, adipokines (such as leptin), and cytokines (such as TNF-α and IL-6) into the portal circulation.
      • Parker R.
      • Kim S.-J.
      • Gao B.
      Alcohol, adipose tissue and liver disease: mechanistic links and clinical considerations.
      ,
      • Shim Y.-R.
      • Jeong W.-I.
      Recent advances of sterile inflammation and inter-organ cross-talk in alcoholic liver disease.
      These adipokines, like leptin, have proinflammatory effects on the liver. Leptin (along with other endocrine factors) activates the HSCs and Kupffer cells (that produce increased TNF-α), and thereby promotes hepatic inflammation and fibrosis (Figure 3). High levels of TNF-α can damage the liver hepatocytes, as discussed previously.
      • Shim Y.-R.
      • Jeong W.-I.
      Recent advances of sterile inflammation and inter-organ cross-talk in alcoholic liver disease.
      Also, leptin and acetaldehyde together can enhance the production of IL-6 in the HSCs (Figure 3).
      • Liu Y.
      • Brymora J.
      • Zhang H.
      • Smith B.
      • Ramezani–Moghadam M.
      • George J.
      • Wang J.
      Leptin and acetaldehyde synergistically promotes [alpha]SMA expression in hepatic stellate cells by an interleukin 6-dependent mechanism.
      Interestingly, leptin levels correlate with liver disease severity in patients with alcoholic cirrhosis.
      • Parker R.
      • Kim S.-J.
      • Gao B.
      Alcohol, adipose tissue and liver disease: mechanistic links and clinical considerations.
      In addition, iron loading in the adipocytes reduces the production of the anti-inflammatory adipokine adiponectin. This can further promote inflammation and contribute to liver injury.
      • Gabrielsen J.S.
      • Gao Y.
      • Simcox J.A.
      • Huang J.
      • Thorup D.
      • Jones D.
      • Cooksey R.C.
      • Gabrielsen D.
      • Adams T.D.
      • Hunt S.C.
      • Hopkins P.N.
      • Cefalu W.T.
      • McClain D.A.
      Adipocyte iron regulates adiponectin and insulin sensitivity.
      Although the aforementioned cellular interactions showcase an iron perspective, ALD pathology is additionally driven by both adaptive and innate immune systems and involves the recruitment of various immune cells that generate a proinflammatory environment in the liver.
      • Li S.
      • Tan H.-Y.
      • Wang N.
      • Feng Y.
      • Wang X.
      • Feng Y.
      Recent insights into the role of immune cells in alcoholic liver disease.
      As such, the liver has abundant lymphocytes scattered through its parenchyma, and it is also rich in cells of the innate immune system, such as the natural killer cells.
      • Seo W.
      • Jeong W.-I.
      Hepatic non-parenchymal cells: master regulators of alcoholic liver disease?.
      Iron does play a role in liver pathology via the immune cells. For example, iron deficiency dampened concanavalin A–induced intrahepatic inflammation in mice. It also reduced intrahepatic lymphocyte infiltration.
      • Bonaccorsi-Riani E.
      • Danger R.
      • Lozano J.J.
      • Martinez-Picola M.
      • Kodela E.
      • Mas-Malavila R.
      • Bruguera M.
      • Collins H.L.
      • Hider R.C.
      • Martinez-Llordella M.
      • Sanchez-Fueyo A.
      Iron deficiency impairs intra-hepatic lymphocyte mediated immune response.

      Low Liver Iron Content: A Phenomenon to Be Investigated

      In a study by Varghese et al,
      • Varghese J.
      • James J.V.
      • Sagi S.
      • Chakraborty S.
      • Sukumaran A.
      • Ramakrishna B.
      • Jacob M.
      Decreased hepatic iron in response to alcohol may contribute to alcohol-induced suppression of hepcidin.
      mice models showed gradual elevation of serum iron levels during 12 weeks of alcohol feeding. Elevations in duodenal ferroportin (gradually increased at 8 weeks and further at 12 weeks) and duodenal DMT1 (significantly increased at 8 weeks but decreased to control levels at 12 weeks) supported this increment in serum iron. In contrast to these elevations, hepatic and serum hepcidin expression gradually decreased during the 12 weeks of alcohol exposure.
      • Varghese J.
      • James J.V.
      • Sagi S.
      • Chakraborty S.
      • Sukumaran A.
      • Ramakrishna B.
      • Jacob M.
      Decreased hepatic iron in response to alcohol may contribute to alcohol-induced suppression of hepcidin.
      This alcohol-induced decrement in hepcidin is an expected response and is also seen in patients with ALD.
      • Bridle K.
      • Cheung T.-K.
      • Murphy T.
      • Walters M.
      • Anderson G.
      • Crawford D.G.
      • Fletcher L.M.
      Hepcidin is down-regulated in alcoholic liver injury: implications for the pathogenesis of alcoholic liver disease.
      • Costa-Matos L.
      • Batista P.
      • Monteiro N.
      • Simões M.
      • Egas C.
      • Pereira J.
      • Pinho H.
      • Santos N.
      • Ribeiro J.
      • Cipriano M.A.
      • Henriques P.
      • Girão F.
      • Rodrigues A.
      • Carvalho A.
      Liver hepcidin mRNA expression is inappropriately low in alcoholic patients compared with healthy controls.
      • Dostalikova-Cimburova M.
      • Balusikova K.
      • Kratka K.
      • Chmelikova J.
      • Hejda V.
      • Hnanicek J.
      • Neubauerova J.
      • Vranova J.
      • Kovar J.
      • Horak J.
      Role of duodenal iron transporters and hepcidin in patients with alcoholic liver disease.
      • Harrison-Findik D.D.
      Is the iron regulatory hormone hepcidin a risk factor for alcoholic liver disease?.
      Herein, the lack of hepcidin up-regulation despite elevation in serum iron levels reiterates the insensitivity of hepcidin to increasing systemic iron levels in the presence of alcohol.
      Distinct from the frequently observed hepatic iron elevation in alcoholics, herein, in mice models, hepatic iron levels were seen to decrease after 12 weeks of alcohol feeding.
      • Varghese J.
      • James J.V.
      • Sagi S.
      • Chakraborty S.
      • Sukumaran A.
      • Ramakrishna B.
      • Jacob M.
      Decreased hepatic iron in response to alcohol may contribute to alcohol-induced suppression of hepcidin.
      The pattern of liver iron decrement matched fully with the patterns of decrements of hepatic TfR1 hepatic ferritin expressions through the 12 weeks of alcohol exposure. This decrease in liver iron content is an unexpected response because several studies in humans have shown increased liver iron content in alcohol consumers/patients with ALD.
      • Eng S.C.
      • Taylor S.L.
      • Reyes V.
      • Raaka S.
      • Berger J.
      • Kowdley K.V.
      Hepatic iron overload in alcoholic end-stage liver disease is associated with iron deposition in other organs in the absence of HFE-1 hemochromatosis.
      • Kohgo Y.
      • Ohtake T.
      • Ikuta K.
      • Suzuki Y.
      • Hosoki Y.
      • Saito H.
      • Kato J.
      Iron accumulation in alcoholic liver diseases.
      • Kowdley K.V.
      Iron overload in patients with chronic liver disease.
      Varghese et al
      • Varghese J.
      • James J.V.
      • Sagi S.
      • Chakraborty S.
      • Sukumaran A.
      • Ramakrishna B.
      • Jacob M.
      Decreased hepatic iron in response to alcohol may contribute to alcohol-induced suppression of hepcidin.
      attributed the decrement in hepcidin expression partly to decreased hepatic iron levels. The authors proposed that this could be due to alcohol-induced hepatomegaly and alcoholic steatosis and/or mobilization of iron to other tissues. The concept of mobilization of iron from liver to other tissues was supported by their observation that hepatic ferroportin expression showed a tendency to increase after 4 and 12 weeks of alcohol exposure, which would facilitate cellular iron egress.
      • Varghese J.
      • James J.V.
      • Sagi S.
      • Chakraborty S.
      • Sukumaran A.
      • Ramakrishna B.
      • Jacob M.
      Decreased hepatic iron in response to alcohol may contribute to alcohol-induced suppression of hepcidin.
      The reason for decrement in liver iron content needs to be fully understood, particularly because it involves the function of ferroportin, the sole known unidirectional cellular iron transporter.

      Liver Iron Loading in ALD: Diagnostic, Prognostic, and Therapeutic Perspectives

      Liver Iron and ALD Diagnosis and Prognosis

      Currently, there is no single diagnostic test to confirm ALD.
      • Torruellas C.
      • French S.W.
      • Medici V.
      Diagnosis of alcoholic liver disease.
      One of the challenges at the diagnostic front is that the symptoms of ALD are not obvious at the early stages. Suspected cases are often tackled based on patient-derived information about their alcohol intake (patient history) supported by laboratory tests. Crabb et al
      • Crabb D.W.
      • Im G.Y.
      • Szabo G.
      • Mellinger J.L.
      • Lucey M.R.
      Diagnosis and treatment of alcohol-associated liver diseases: 2019 practice guidance from the American Association for the Study of Liver Diseases.
      have reviewed this topic in detail. Note that liver iron loading by itself cannot be used for the diagnosis of ALD or any chronic liver disease because there are several other liver conditions, such as hemochromatosis and nonalcoholic fatty liver disease, that show high liver iron content.
      • Mehta K.J.
      • Farnaud S.J.
      • Sharp P.A.
      Iron and liver fibrosis: mechanistic and clinical aspects.
      An old study indicated that liver iron in ALD has a prognostic value. It showed that patients with alcoholic cirrhosis with detectable liver iron had a lower survival rate than those without.
      • Ganne-Carrié N.
      • Christidis C.
      • Chastang C.
      • Ziol M.
      • Chapel F.
      • Imbert-Bismut F.
      • Trinchet J.C.
      • Guettier C.
      • Beaugrand M.
      Liver iron is predictive of death in alcoholic cirrhosis: a multivariate study of 229 consecutive patients with alcoholic and/or hepatitis C virus cirrhosis: a prospective follow up study.
      However, other authors suggested that hepatic iron overload is a poor prognostic factor in ALD.
      • Whitfield J.B.
      • Zhu G.
      • Heath A.C.
      • Powell L.W.
      • Martin N.G.
      Effects of alcohol consumption on indices of iron stores and of iron stores on alcohol intake markers.

      Liver Iron and ALD Therapeutics: Alcohol Abstinence

      Although there are US Food and Drug Administration–approved therapies for alcohol use disorders that help reduce cravings for alcohol,
      • Crabb D.W.
      • Im G.Y.
      • Szabo G.
      • Mellinger J.L.
      • Lucey M.R.
      Diagnosis and treatment of alcohol-associated liver diseases: 2019 practice guidance from the American Association for the Study of Liver Diseases.
      ,
      • Singal A.K.
      • Mathurin P.
      Diagnosis and treatment of alcohol-associated liver disease: a review.
      there is no US Food and Drug Administration–approved drug to treat ALD.
      • Wong V.W.-S.
      • Singal A.K.
      Emerging medical therapies for non-alcoholic fatty liver disease and for alcoholic hepatitis.
      Alcohol abstinence is the only curative option, and liver transplantation is the definitive treatment for liver diseases (including ALD) in the end stage.
      Cessation of alcohol has shown to reduce liver iron deposits. For example, a study found that patients with ALD who abstained for >3 months had reduced liver iron content compared with patients with ALD with active alcoholism (average intake of 164.4 g/day).
      • Costa Matos L.
      • Batista P.
      • Monteiro N.
      • Ribeiro J.
      • Cipriano M.A.
      • Henriques P.
      • Girão F.
      • Carvalho A.
      Iron stores assessment in alcoholic liver disease.
      Also, drinking lesser amount of alcohol has shown to cause lesser liver iron deposition. For example, in a study, mean liver iron concentrations were significantly higher in alcoholic patients (who drank >80 g/day for ≥3 years before and inclusive of the study period) compared with controls who did not drink excessive amounts of alcohol (ie, did not drink >20 g/day).
      • Chapman R.W.
      • Morgan M.Y.
      • Laulicht M.
      • Hoffbrand A.V.
      • Sherlock S.
      Hepatic iron stores and markers of iron overload in alcoholics and patients with idiopathic hemochromatosis.

      Liver Iron and ALD Therapeutics: Discussing Phlebotomy

      Hemochromatosis is an iron-overload disease in which patients show high systemic and liver iron loading, in addition to iron deposition in other organs.
      • Brissot P.
      • Pietrangelo A.
      • Adams P.C.
      • de Graaff B.
      • McLaren C.E.
      • Loréal O.
      Haemochromatosis.
      For patients with hemochromatosis who show high iron loading, life-long periodic phlebotomy is the mainstay of therapy, where the aim is to reduce the level of iron and thereby limit excess iron–induced organ damage. In a patient with ferroprotein disease (hereditary iron loading disorder), long-term phlebotomy decreased hepatic iron accumulation.
      • Nishina S.
      • Tomiyama Y.
      • Ikuta K.
      • Tatsumi Y.
      • Toki Y.
      • Kato A.
      • Kato K.
      • Yoshioka N.
      • Sasaki K.
      • Hara Y.
      • Hino K.
      Long-term phlebotomy successfully alleviated hepatic iron accumulation in a ferroportin disease patient with a mutation in SLC40A1: a case report.
      This questions whether phlebotomy could be used for patients with ALD who show liver iron overload. First, just like in case of hemochromatosis, where not all patients demonstrate enough iron overload to cause organ damage,
      • Brissot P.
      • Pietrangelo A.
      • Adams P.C.
      • de Graaff B.
      • McLaren C.E.
      • Loréal O.
      Haemochromatosis.
      ,
      • Palmer W.C.
      • Vishnu P.
      • Sanchez W.
      • Aqel B.
      • Riegert-Johnson D.
      • Seaman L.A.K.
      • Bowman A.W.
      • Rivera C.E.
      Diagnosis and management of genetic iron overload disorders.
      not all patients with ALD show liver iron loading.
      • Mueller S.
      • Rausch V.
      The role of iron in alcohol-mediated hepatocarcinogenesis.
      ,
      • Varghese J.
      • James J.V.
      • Sagi S.
      • Chakraborty S.
      • Sukumaran A.
      • Ramakrishna B.
      • Jacob M.
      Decreased hepatic iron in response to alcohol may contribute to alcohol-induced suppression of hepcidin.
      ,
      • Milic S.
      • Mikolasevic I.
      • Orlic L.
      • Devcic E.
      • Starcevic-Cizmarevic N.
      • Stimac D.
      • Kapovic M.
      • Ristic S.
      The role of iron and iron overload in chronic liver disease.
      Some patients with ALD may be anemic.
      • Scheiner B.
      • Semmler G.
      • Maurer F.
      • Schwabl P.
      • Bucsics T.A.
      • Paternostro R.
      • Bauer D.
      • Simbrunner B.
      • Trauner M.
      • Mandorfer M.
      • Reiberger T.
      Prevalence of and risk factors for anaemia in patients with advanced chronic liver disease.
      Second, in patients with ALD who show liver iron loading, the levels hardly ever surpass two to three times the upper limit of the norm.
      • Batts K.P.
      Iron overload syndromes and the liver.
      Third, phlebotomy has several limitations, one of which is the possibility of developing anemia.
      • Milic S.
      • Mikolasevic I.
      • Orlic L.
      • Devcic E.
      • Starcevic-Cizmarevic N.
      • Stimac D.
      • Kapovic M.
      • Ristic S.
      The role of iron and iron overload in chronic liver disease.
      Therefore, although phlebotomy is a suitable option for iron-overloaded patients with hemochromatosis, it is not a suitable therapeutic option for patients with ALD.

      Liver Iron and ALD Therapeutics: Iron Chelation

      In general, apart from phlebotomy, another therapeutic approach for reducing liver iron content is iron chelation by using iron chelators like deferoxamine, deferiprone, and deferasirox.
      • Aydinok Y.
      • Kattamis A.
      • Cappellini M.D.
      • El-Beshlawy A.
      • Origa R.
      • Elalfy M.
      • Kilinç Y.
      • Perrotta S.
      • Karakas Z.
      • Viprakasit V.
      • Habr D.
      • Constantinovici N.
      • Shen J.
      • Porter J.B.
      on behalf of the HYPERION Investigators
      Effects of deferasirox-deferoxamine on myocardial and liver iron in patients with severe transfusional iron overload.
      Deferiprone
      • Mobarra N.
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      A review on iron chelators in treatment of iron overload syndromes.
      Deferiprone decreased hepatocyte iron overload in chronically ethanol-fed rats.
      • Sadrzadeh S.M.
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      • Price P.L.
      The oral iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one reduces hepatic-free iron, lipid peroxidation and fat accumulation in chronically ethanol-fed rats.
      A novel iron chelator, M30, reduced alcohol-indued injury in rat hepatocytes. There was a significant attenuation of ethanol-induced apoptosis, oxidative stress, and secretion of inflammatory cytokines.
      • Xiao J.
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      A novel antioxidant multitarget iron chelator M30 protects hepatocytes against ethanol-induced injury.
      Thus, these chelators could be tried for ALD cases that show liver iron overload.
      Naturally occurring compounds (namely, flavonoids) are also potential therapeutic agents. These have shown to impair ALD pathologic progression by maintaining iron balance. For example, quercetin, which exhibits iron-chelating and antioxidant properties, dampened alcohol-induced liver damage in mice.
      • Li L.-X.
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      Iron overload in alcoholic liver disease: underlying mechanisms, detrimental effects, and potential therapeutic targets.
      Such natural compounds could be tested in alcohol-treated animal models and then relevant clinical trials could be established.

      Liver Iron and ALD Therapeutics: Synthetic Hepcidin

      Hepcidin deficiency is the main cause of iron loading in patients with ALD.
      • Bridle K.
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      • Walters M.
      • Anderson G.
      • Crawford D.G.
      • Fletcher L.M.
      Hepcidin is down-regulated in alcoholic liver injury: implications for the pathogenesis of alcoholic liver disease.
      ,
      • Harrison-Findik D.D.
      • Schafer D.
      • Klein E.
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      • Kulaksiz H.
      • Clemens D.
      • Fein E.
      • Andriopoulos B.
      • Pantopoulos K.
      • Gollan J.
      Alcohol metabolism-mediated oxidative stress down-regulates hepcidin transcription and leads to increased duodenal iron transporter expression.
      Therefore, hepcidin treatment is a promising therapeutic approach. Natural hepcidin is expensive and has undesirable pharmacologic properties, such as having a short half-life. In contrast, minihepcidins are synthetic in nature. These mimic the action of hepcidin and are pharmacologically more favorable.
      • Milic S.
      • Mikolasevic I.
      • Orlic L.
      • Devcic E.
      • Starcevic-Cizmarevic N.
      • Stimac D.
      • Kapovic M.
      • Ristic S.
      The role of iron and iron overload in chronic liver disease.
      I.p. injections of minihepcidin to mice models of hemochromatosis showed significant reductions in liver iron loading.
      • Preza G.C.
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      Minihepcidins are rationally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overload.
      This experiment could be repeated in alcohol-fed animal models to extrapolate whether the approach would be successful in reducing liver iron loading in patients with ALD.

      Liver Iron and ALD Therapeutics: Targeting Ferroptosis

      Interestingly, not the liver iron loading itself, but ferroptosis, the iron-dependent process that contributes to liver damage in ALD, has been targeted for therapy. Ferroptosis inhibitors and repressors have shown protective effects against alcohol-induced liver damage. For example, the ferroptosis inhibitor ferrostatin-1 reduced lipid peroxidation and alcohol-induced liver injury in vivo.
      • Liu C.-Y.
      • Wang M.
      • Yu H.-M.
      • Han F.-X.
      • Wu Q.-S.
      • Cai X.-J.
      • Kurihara H.
      • Chen Y.-X.
      • Li Y.-F.
      • He R.-R.
      Ferroptosis is involved in alcohol-induced cell death in vivo and in vitro.
      Similarly, another ferroptosis inhibitor, dimethyl fumarate, significantly improved alcohol-induced liver injury in ethanol-fed mice.
      • Zhang Y.
      • Zhao S.
      • Fu Y.
      • Yan L.
      • Feng Y.
      • Chen Y.
      • Wu Y.
      • Deng Y.
      • Zhang G.
      • Chen Z.
      • Chen Y.
      • Liu T.
      Computational repositioning of dimethyl fumarate for treating alcoholic liver disease.
      Also, deficiency of intestinal SIRT1 in mice has shown protection from alcohol-induced hepatic injury via mitigation of ferroptosis.
      • Zhou Z.
      • Ye T.J.
      • DeCaro E.
      • Buehler B.
      • Stahl Z.
      • Bonavita G.
      • Daniels M.
      • You M.
      Intestinal SIRT1 deficiency protects mice from ethanol-induced liver injury by mitigating ferroptosis.
      Frataxin is a mitochondrial protein that predominantly participates in iron homeostasis and oxidative stress. A study showed that alcohol reduced the expression of frataxin, and the deficiency of frataxin increased sensitivity to alcohol-induced ferroptosis (Figure 2). Restoration of frataxin reversed this effect.
      • Liu J.
      • He H.
      • Wang J.
      • Guo X.
      • Lin H.
      • Chen H.
      • Jiang C.
      • Chen L.
      • Yao P.
      • Tang Y.
      Oxidative stress-dependent frataxin inhibition mediated alcoholic hepatocytotoxicity through ferroptosis.
      Thus, frataxin can be an additional therapeutic target to tackle ALD.

      Summary

      Increased serum iron due to chronic alcohol consumption increases iron uptake in the hepatocytes and Kupffer cells, facilitating both parenchymal and nonparenchymal iron loading in the liver, and in parenchymal cells of other organs. Hepatic iron deposition is mediated via up-regulation of TfR1 and HFE (proposed). Both iron and alcohol can independently induce oxidative stress, so the combined effect accelerates hepatic injury. Excess iron-simulated hepatocytes and Kupffer cells secrete inflammatory and profibrogenic factors that activate the hepatic stellate cells. Chronic activation of hepatic stellate cells mediates the development of liver fibrosis. Iron loading promotes ALD progression via induction of oxidative stress and the activation of HSCs and Kupffer cells. Other cells, such as the liver sinusoidal endothelial cells, the liver immune cells (from both adaptive and innate immune systems), and the adipocytes, also contribute to the iron-mediated liver injury in ALD.

      Author Contributions

      K.F. and N.A. performed the primary investigation and wrote the original draft; and K.J.M. conceptualized and supervised the study, and wrrote, and edited the manuscript.

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