Mitochondrial Dysfunction: At the Nexus between Alcohol-Associated Immunometabolic Dysregulation and Tissue Injury

Round 1

Reviewer 1 Report

Siggins, et al. provide a concise and succinct overview of the effects of alcohol on mitochondrial function and its relationship to both intrinsic and extrinsic immunometabolism.  This review is very well organized and would serve as a good overview for both new and experienced investigators in this area of research.  However, the completeness of this review and its connection to long-term disease could be improved upon to further its utility. 

General Comments: 

1. When discussing the tissue damage associated with alcohol misuse it would be beneficial to identify the mechanism(s) of cell death that have been implicated.   

2. Throughout the review, the authors quote "ROS production/generation" associated caused by alcohol misuse.  The specific type of ROS generated (e.g., superoxide, hydrogen peroxide) should be mentioned to give context as "ROS" is a general term.  Additionally, it may be helpful to provide some information regarding the method(s) of ROS detection that was used to make these claims.    

3.  Some background information should be provided regarding the pathophysiology associated with mitochondrial damage/ROS production resulting in diseased tissue.  How do these metabolic changes affect the onset of fibrosis and subsequent disease (e.g., cirrhosis)?  Any additional evidence of redox modulation of diseased states (e.g., effects of superoxide dismutase and other antioxidants on liver disease, PMID: 11477087 PMID: 31928222, PMID:22093324) would help explicitly connect the biochemical changes to translational approaches.

1. Is there any evidence that alcohol misuse disrupts iron metabolism?  Iron is a critical redox-active metal that serves as a catalyst in many ROS-regulated processes/diseases.  This should be mentioned. 

Minor comments: 

1. In section "2.1 Liver" lines 146-150, the authors describe the effects of alcohol withdrawal on the expression of several key metabolic proteins.  Can this be connected to altered Nrf2 expression? Many of these enzymes would have an antioxidant response element in their promoter region. 

2.  Macrophages have very high intracellular iron concentrations that are central to their function.  What changes in iron content/metabolism occur during the polarization process?  

Author Response

Reviewer 1. The completeness of this review and its connection to long-term disease could be improved upon to further its utility.

Response: We appreciate the constructive feedback, and in addition to the specific concerns raised by the reviewer, we have added further discussion of alcohol-mediated mitochondrial dysfunction as a driver for long term disease on lines 594-611.

In addition to ATP generation, metabolism participates in a variety of cellular processes that impact tissue crosstalk. Numerous lines of evidence support mitochondrial dysfunction preceding many non-communicable diseases, which has been reviewed recently (Diaz-Vegas, Sanchez-Aguilera et al, 2020). While progressive mitochondrial damage may lead to further tissue functional impairment and long-term disease progression, there is evidence that interventions, such as caloric restriction and exercise, enhance mitochondrial function and ameliorate some of the deficits of chronic diseases, like chronic in-flammation obesity, and type 2 diabetes mellitus (T2DM) (Gabandé-Rodríguez , Gómez de Las Heras et al 2019; Kim, Wei et al, 2008; Krako Jakovljevic , Pavlovic K et al, 2021; ). Clinical and preclinical studies show that alcohol impairs mitochondrial function (Hoek, Cahill et al. 2002). There is compelling evidence that chronic alcohol consumption medi-ates mitochondrial damage leading to the progressive development of T2DM (Kim, Lee et al 2015). A strong link between alcohol-induced mitochondrial damage underpinning disease pathogenesis was elucidated recently in a study showing that hepatocytes from ARLD patients are selectively depleted in MATα1, and preserving mitochondrial MATα1 content prevented alcohol-induced mitochondrial dysfunction (Barbier-Torres, Murray et al, 2022).

1. When discussing the tissue damage associated with alcohol misuse it would be beneficial to identify the mechanism(s) of cell death that have been implicated.   

Response: Thank you for the suggestion. We have incorporated the mechanism of cell death when the original literature was specified. We have edited lines 89 and 104 as follows.

Line 89à “Central to these processes is mitochondrial function, and alcohol-mediated mitochondrial dysfunction has been studied extensively in alcohol-mediated liver injury ultimately causing hepatocyte apoptosis, necroptosis, or necrosis.”

Line 104à “In PPARα knock out mice fed 4% alcohol, there was increased hepatic injury including mitochondrial swelling, oxidative stress, inflammation, fibrosis, and apoptosis.”

2. Throughout the review, the authors quote "ROS production/generation" caused by alcohol misuse.  The specific type of ROS generated (e.g., superoxide, hydrogen peroxide) should be mentioned to give context as "ROS" is a general term.  Additionally, it may be helpful to provide some information regarding the method(s) of ROS detection that was used to make these claims.    

Response: We agree with the reviewer that ROS may be too general a term. What is particularly confounding is that very few reports utilize the gold standard for ROS detection, which is electron paramagnetic resonance (EPR) spectrometry. Therefore, it is difficult to state with certainty which ROS is being produced under most circumstances. More often than not, studies will provide measurements of H2O2, which is a longer-lasting product derived from superoxide. Due to this limitation in accurate measures in the published literature, we opted to include a statement about mitochondrial ROS with a citation for a recent review on ROS signaling on lines 49-55.

“Mitochondria produce mostly superoxide anion and hydrogen peroxide, and these molecules play important roles in immune cell function and dysfunction, which has recently been reviewed (Bassoy, Walch et al, 2021). Additionally, there is also compelling evidence that increased ROS leads to tissue injury, such as hepatic fibrogenesis (Ceni, Mello et al, 2014, Singal, Jampana et al, 2011). However, alcohol-mediated mitochondrial ROS generation and the mechanisms that lead to tissue injury are beyond the scope of this review.”

14. Bassoy EY, Walch M, Martinvalet D. Reactive Oxygen Species: Do They Play a Role in Adaptive Immunity? Front Immunol. 2021 Nov 22;12:755856. doi: 10.3389/fimmu.2021.755856. PMID: 34899706; PMCID: PMC8653250.
15. Ceni E, Mello T, Galli A. Pathogenesis of alcoholic liver disease: role of oxidative metabolism. World J Gastroenterol. 2014 Dec 21;20(47):17756-72. doi: 10.3748/wjg.v20.i47.17756. PMID: 25548474; PMCID: PMC4273126.
16. Singal AK, Jampana SC, Weinman SA. Antioxidants as therapeutic agents for liver disease. Liver Int. 2011 Nov;31(10):1432-48. doi: 10.1111/j.1478-3231.2011.02604.x. Epub 2011 Jul 29. PMID: 22093324; PMCID: PMC3228367.
17. Some background information should be provided regarding the pathophysiology associated with mitochondrial damage/ROS production resulting in diseased tissue. How do these metabolic changes affect the onset of fibrosis and subsequent disease (e.g., cirrhosis)? Any additional evidence of redox modulation of diseased states (e.g., effects of superoxide dismutase and other antioxidants on liver disease,PMID:11477087, PMID:31928222, PMID:22093324) would help explicitly connect the biochemical changes to translational approaches.

Response: We have added more clarification as suggested regarding the pathophysiology associated with mitochondrial damage/ROS production, as outlined below. However, the review focuses on immunometabolic alterations, and discussing the specific mechanisms of alcohol-mediated mitochondrial ROS generation leading to tissue injury are beyond the scope of this review.

Lines 52-54. Additionally, there is also compelling evidence that increased ROS leads to tissue injury such as hepatic fibrogenesis (Ceni, Mello et al, 2014, Singal, Jampana et al, 2011).

Lines 154-160à “In a rat model of chronic alcohol feeding, steatosis, inflammation, and necrosis were all decreased in animals receiving adenoviral vectors containing a mitochondrial-targeted manganese superoxide dismutase (Mn-SOD; Wheeler, Nakagami et al, 2001). In a similar study of chronic ethanol feeding in the setting of a high fat diet, nanoparticle delivery of SOD1 was able to prevent alcohol-induced liver damage, partially through downregulation of cytochrome P450 (CYP)2E1 (Gopal et al. 2020).”

24. Ceni E, Mello T, Galli A. Pathogenesis of alcoholic liver disease: role of oxidative metabolism. World J Gastroenterol. 2014 Dec 21;20(47):17756-72. doi: 10.3748/wjg.v20.i47.17756. PMID: 25548474; PMCID: PMC4273126.
25. Gopal T, Kumar N, Perriotte-Olson C, Casey CA, Donohue TM Jr, Harris EN, Talmon G, Kabanov AV, Saraswathi V. Nanoformulated SOD1 ameliorates the combined NASH and alcohol-associated liver disease partly via regulating CYP2E1 expression in adipose tissue and liver. Am J Physiol Gastrointest Liver Physiol. 2020 Mar 1;318(3):G428-G438. doi: 10.1152/ajpgi.00217.2019. Epub 2020 Jan 13. PMID: 31928222; PMCID: PMC7099493.
26. Singal AK, Jampana SC, Weinman SA. Antioxidants as therapeutic agents for liver disease. Liver Int. 2011 Nov;31(10):1432-48. doi: 10.1111/j.1478-3231.2011.02604.x. Epub 2011 Jul 29. PMID: 22093324; PMCID: PMC3228367.
27. Wheeler MD, Nakagami M, Bradford BU, Uesugi T, Mason RP, Connor HD, Dikalova A, Kadiiska M, Thurman RG. Overex-pression of manganese superoxide dismutase prevents alcohol-induced liver injury in the rat. J Biol Chem. 2001 Sep 28;276(39):36664-72. doi: 10.1074/jbc.M105352200. Epub 2001 Jul 26. PMID: 11477087.
28. Is there any evidence that alcohol misuse disrupts iron metabolism?  Iron is a critical redox-active metal that serves as a catalyst in many ROS-regulated processes/diseases.  This should be mentioned. 

Response: There is literature on the impact of alcohol use and iron metabolism. We have added brief additional information in the introduction. We agree that dysregulated iron metabolism is a potential contributor to alcohol-mediated increased ROS, however, as we have focused mainly on alcohol-mediated immunometabolism, the review does not elaborate on these mechanisms:

Lines 38-41à “Multiple signaling pathways are disrupted in these tissues, including dysregulated iron metabolism that can potentially increase oxidative stress, and this disruption is dependent on the quantity, frequency, and duration of alcohol intake (Farrao, Ali et al, 2022, Harrison-Findik, 2007, Molina, Gardner et al 2014).”

47. Ferrao K, Ali N, Mehta KJ. Iron and iron-related proteins in alcohol consumers: cellular and clinical aspects. J Mol Med (Berl). 2022 Dec;100(12):1673-1689. doi: 10.1007/s00109-022-02254-8. Epub 2022 Oct 10. PMID: 36214835; PMCID: PMC9691479.
48. Harrison-Findik DD. Role of alcohol in the regulation of iron metabolism. World J Gastroenterol. 2007 Oct 7;13(37):4925-30. doi: 10.3748/wjg.v13.i37.4925. PMID: 17854133; PMCID: PMC4434614.

Minor comments: 

1. In section "2.1 Liver" lines 146-150, the authors describe the effects of alcohol withdrawal on the expression of several key metabolic proteins.  Can this be connected to altered Nrf2 expression? Many of these enzymes would have an antioxidant response element in their promoter region. 

Response: We appreciate the suggestion and have updated the text and included an additional reference.

Lines 165-168à  “Ethanol withdrawal has also been shown to increase an important antioxidant pathway transcription factor, nuclear respiratory factor 2 (Nrf2), within 1 week of alcohol cessation in a binge-on-chronic mouse model (Kang, Kim et al. 2022).”

78. Kang H, Kim MB, Park YK, Lee JY. A mouse model of the regression of alcoholic hepatitis: Monitoring the regression of hepatic steatosis, inflammation, oxidative stress, and NAD+ metabolism upon alcohol withdrawal. J Nutr Biochem. 2022 Jan;99:108852. doi: 10.1016/j.jnutbio.2021.108852. Epub 2021 Sep 12. PMID: 34525389.
79. Macrophages have very high intracellular iron concentrations that are central to their function.  What changes in iron content/metabolism occur during the polarization process?  

Response: This is a good point, and we have updated the text and included an additional reference. Though there is literature to support the role of iron on macrophage polarization, the link the link between iron and macrophage polarization with alcohol use is less clear. We have added the following paragraph to lines 471-479:

“Intracellular iron concentrations are important to macrophage polarization processes. It has been shown that low iron increases pro-inflammatory M1, likely through Krebs cycle inhibition, and high iron increases anti-inflammatory M2 macrophages (Agoro, Taleb et al, 2018). However, the link between iron and macrophage polarization with alcohol use is less clear. Alcohol increases hepatic and circulating iron levels in ARLD (Xiong, She, et al 2008) Kupffer cells increase their iron uptake, but rather than transitioning to an M2 phenotype, they seem to remain pro-inflammatory. This indicates that iron status and alcohol are potentially having independent effects on mitochondrial bioenergetics and macrophage polarization.”

Reviewer 2 Report

In their paper, Siggins et al. summarize the current state of knowledge on alcohol-related tissue damage. Special attention was given to mitochondrial dysregulation in various organs and cells of the immune system. The work is very well elaborated. However, there are a lot of abbreviations used in the text. I suggest creating a separate section for abbreviations. The last paragraph devoted to discussion should be more of a conclusion. After a small revision, the paper can be accepted for publication.

Author Response

Reviewer 2.

1. There are a lot of abbreviations used in the text. I suggest creating a separate section for abbreviations.

Response: We have added an abbreviations list to lines 661-687.

2. The last paragraph devoted to discussion should be more of a conclusion. After a small revision, the paper can be accepted for publication.

Response: We have added a conclusion paragraph to lines 642-651:

“5. Conclusion

Alcohol misuse causes deleterious effects in every tissue. However, there are no single unifying mechanisms for alcohol’s pathological effects. Because mitochondrial function under healthy physiological conditions is tailored to tissue metabolic needs, it only stands to reason that alcohol-impaired mitochondrial function is a critical contributor to tissue injury. As discussed in this review, mitochondrial damage is a key effector of metabolic tissue dysfunction, and mitochondrial dysfunction underlies considerable dysregulation of immune function through aberrant intrinsic and extrinsic immunometabolism. The pervasive damaging effects of alcohol, in part, can thus be attributed to the detrimental crosstalk between impaired metabolic tissue signaling and immune cell function, with mitochondrial dysfunction at the nexus of this interaction.”

Reviewer 3 Report

In this literature review ‘Mitochondrial dysfunction: At the nexus between alcohol-associated immunometabolic dysregulation and tissue injury’ (IJMS-2362500), Siggins et al. do a commendable job of putting together a great deal of literature. They discuss clearly the nuances of the harmful effects of the most widely abused substance in society, i.e., alcohol on several tissues and cell types, paying particular attention to mitochondria. The review is well-balanced and succinct.

The present version needs only minor revision to improve its readability. This revision concerns all the three figures, which as of now contain just titles. It will be great if these figures have a brief legend describing the graphics.

Author Response

Reviewer 3.

This revision concerns all the three figures, which as of now contain just titles. It will be great if these figures have a brief legend describing the graphics.

Response: We have updated the figure legends within the manuscript.

Lines 324-331à “Figure 1. Alcohol-mediated mitochondrial dysregulation in metabolic regulatory tissues. Alcohol (EtOH) impairs electron transport chain (ETC) function, mitochondrial fusion/fission, and mitophagy, while promoting apoptosis, increasing mitochondrial mass and reactive oxygen species, and immune cell infiltration in the illustrated tissues. The effects of alcohol on mitochondria are shown in purple and tissue signaling outcomes are shown in maroon in each box corresponding to the individual tissues.

Lines 445-452à “Figure 2. Alcohol-mediated mitochondrial dysregulation contributes to pro-inflammatory environment. Chronic alcohol (EtOH) use impairs mitochondrial function and repair in immune cells and decreases neutrophil, CD8+ T cells, and B cells and increases pro-inflammatory M1 macrophage and Th1 CD4+ T cells. The effects of alcohol on mitochondria are shown in purple and immune outcomes are shown in maroon in each box corresponding to the individual immune cells.”

Lines 621-626à “Figure 3. Alcohol-mediated mitochondrial dysfunction is at the nexus of extrinsic and intrinsic immunometabolic adaptations. Ethanol-mediated impairment of mitochondrial function increases metabolic dysfunction in tissue, which dysregulates intrinsic

immunometabolism, leading to immunometabolism dysfunction and thus dysregulated extrinsic immunometabolism with alcohol misuse.”

Reviewer 4 Report

Overall, this seems to be a potentially useful review of an important field. However, the section on the pancreas is not well balanced and in certain respects actually misleading. Clearly the authors know the liver literature much better than the one on the pancreas.

The authors seem to indicate at the very beginning of the section that the actions of alcohol on the pancreas are mediated equally by acetaldehyde and fatty acid ethyl esters. This is questionable. There is a huge literature, dating back to the important paper by Laposata & Lange in Science (231:497-499, 1986) showing that the damaging effect of alcohol on the exocrine pancreas is largely due to the effects of fatty acid ethyl esters, produced as a result of the non-oxidative combination of ethanol and long chain fatty acids. This has recently been comprehensively reviewed (Physiol Rev 101:1691-1744, 2021) and it would actually improve the ms, if ref. 33 (a short and rather old review in a somewhat obscure journal) were to be replaced by a reference to the 2021 Physiol Rev article. The one and only argument for an important role of acetaldehyde that is quoted by the authors is contained in ref. 168. However, the experiments reported in ref 168 that seem to support the authors’ view that oxidative metabolism is the key aspect of pancreatic alcohol toxicity are indirect and conducted on permeabilized cells, using ethanol concentrations that are in excess of the blood levels seen in ‘trained’ alcoholics after even extreme alcohol intake. It would be helpful to readers of this review if the authors were to take into account the ‘real life’ human pathophysiological aspects concerning levels of ethanol and fatty acid ethyl esters, summarized on p.1712 in the above mentioned Physiol Rev article.

Author Response

The section on the pancreas is not well balanced and in certain respects actually misleading. Clearly the authors know the liver literature much better than the one on the pancreas.

The authors seem to indicate at the very beginning of the section that the actions of alcohol on the pancreas are mediated equally by acetaldehyde and fatty acid ethyl esters. This is questionable. There is a huge literature, dating back to the important paper by Laposata & Lange in Science (231:497-499, 1986) showing that the damaging effect of alcohol on the exocrine pancreas is largely due to the effects of fatty acid ethyl esters, produced as a result of the non-oxidative combination of ethanol and long chain fatty acids. This has recently been comprehensively reviewed (Physiol Rev 101:1691-1744, 2021) and it would actually improve the ms, if ref. 33 (a short and rather old review in a somewhat obscure journal) were to be replaced by a reference to the 2021 Physiol Rev article. The one and only argument for an important role of acetaldehyde that is quoted by the authors is contained in ref. 168. However, the experiments reported in ref 168 that seem to support the authors’ view that oxidative metabolism is the key aspect of pancreatic alcohol toxicity are indirect and conducted on permeabilized cells, using ethanol concentrations that are in excess of the blood levels seen in ‘trained’ alcoholics after even extreme alcohol intake. It would be helpful to readers of this review if the authors were to take into account the ‘real life’ human pathophysiological aspects concerning levels of ethanol and fatty acid ethyl esters, summarized on p.1712 in the above mentioned Physiol Rev article.

Response: We agree with the reviewer that the main action of alcohol on pancreas is via fatty acid ethyl esters. We apologize that it read that both acetaldehyde and FAEE equally contribute to the effects. We discussed most of the pancreas effects with FAEEs and cited Dr. Ole Petersen’s work. We have now changed the text to reflect that the major metabolite for alcohol effects on the pancreas is FAEEs. 

Lines 187-192à “The non-oxidative alcohol metabolite fatty acid ethyl esters (FAEEs) are major contributors to alcohol-related pancreatitis and increase mitochondrial dysfunction, decrease ATP production, and impair mitochondrial bioenergetic measures in the pancreas (Criddle, Murphy et al. 2006, Peterson, Gerasimenko et al. 2021, , Petersen, Tepikin et al. 2009, Huang, Booth et al. 2014, Mukherjee, Mareninova et al. 2016).”

Round 2

Reviewer 4 Report

The revision has resulted in an overall improved ms. The pancreatic section which was very misleading in the original version is now more balanced. It is nevertheless a pity that the authors did not take up several of the suggestions in my original report that would have markedly improved the pancreatic section, but just made a rather minimalistic revision by removing erroneous statements.  They have added a more up-to-date and comprehensive reference, which helps.