A variety of murine models of alcohol consumptions are frequently used in preclinical investigations to explore the mechanisms responsible of the immunological effects of alcohol and subsequently used to test novel treatments of ALD41,42.
We aimed to assess hepatic phenotype profiles at cellular immune, transcript, and histological levels in the two most common models of chronic alcohol consumption: MC model and isocaloric LD diet. While MC model mice are receiving regular chow diet, LD diet mice were fed with a liquid diet enriched in lipids. This study documented that interpretation of histological, cellular immune and transcriptome data obtained in murine models should strongly consider the type of model used. Moreover, when we compared gene expression similarities between these murine models of alcohol exposure and human alcoholic hepatitis data from the public domain we found very limited specific shared changes.
Our first observation was that at the histological level, there is more hepatic steatosis in mice on LD with or without alcohol for only 4 weeks compared with the mice exposed on standard 12 weeks MC. This is anticipated considering the contribution of fat to overall caloric intake and supports the concept of augmentation of steatosis by combination of dietary fat and alcohol. Histological effect of LD diet seems to have some correlation with the amount of injury. Although there is a trend toward an increase liver injury assessed by liver enzymes in mice in MC model, addition of alcohol increases AST compared with paired control only in LD diet.
Chronic models of alcohol consumption in mice are characterized by minimal to none inflammation42. As expected, in both models histological grading of inflammation does not depict any significant effect of alcohol when compared with control mice (overall median inflammation severity less than 1). Absence of any alcohol specific effect on overall number of hematopoietic derived cells in the liver is confirmed after the isolations and quantification of total hepatic CD45+ cells in both models.
Completely unexpectedly, there were three times less hepatic CD45+ cells in mice on LD diet when compared with the MC model. Interestingly, it is anticipated to have a significant number of myeloid cells in the murine liver, when we looked into the composition of the hepatic CD45+ cells in MC and LD diet, around 80% of all CD45+ cells belong to the lymphoid lineage (NK cells, B cells, CD3+ cells).
Neutrophils are frequently encountered as a part of histological features of alcohol liver injury in humans36. Alcohol does not significantly increase the number of hepatic neutrophils in the liver of mice on either diet, in spite of an increasing trend in the MC model. Furthermore, mice on the LD diet (both control and alcohol) have less numbers of neutrophils compared with MC mice. Moreover, alcohol exposure for 4 weeks in this model (LD EtOH) is associated with an unexpected trend toward a decrease of the number of neutrophils compared to control (LD Control). This suppressive effect of LD diet on neutrophil populations can be explained and additionally explored in the future by potentially complex mechanisms involving neutrophilic bone marrow development and egress, endothelial adhesion molecules dependent, and cytokine gradient hepatic recruitment, or decreased survival of neutrophils in the pro-inflammatory hepatic environment with steatosis.
Monocytes are another type of innate cells considered to be important in pathogenesis of alcoholic liver injury in humans and potentially cellular targets for new drugs43,44,45. Similar with the neutrophils, LD has a suppressive effect on absolute monocyte counts, independent of the alcohol exposure. Interestingly enough, alcohol has a specific effect on monocytes frequency and amount only in the liver of mice in MC model. This difference clearly supports the concept that MC model may be used to explore alcohol-specific monocyte effect targeted drugs.
Similar with cellular immunological changes, limited amount of common shared abnormalities are observed between the two models of murine alcohol exposure at the transcript levels. In contrast to human data where Ccl2, Ccl3, Ccl20 and Cxcl10 transcripts were significantly changed in alcoholic hepatitis compared to healthy control, none of these statistically significant changes were found in the murine models. A possible explanation is that murine models have only limited exposure to alcohol when compared with humans while the other explanation is that severity of the inflammation is only limited and very early upon alcohol toxicity in murine models. The predominant increase in the lymphocyte recruitment chemokine gradient in mice compared with myeloid in humans raises the question of species specificity of the mechanism involved in alcohol induced inflammation. This comparison of two murine models of chronic alcohol exposure with human data present in public domain has to take into account also the limitation of severity of liver disease, sex distribution, and age of patients included in the study. The human samples used for generation of transcriptomic data belongs to adult patients (mean of 49 years old), more than half males, majority with severe alcoholic hepatitis (77.5%) and cirrhosis (60%), with alcohol intake of 107 g/day40 while in our murine studies samples belong to relatively young female mice, with mild liver tissue injury (only steatosis) and average of alcohol consumption between 16–21 g/Kg/day.
Consistent with our findings from RT-qPCR of liver tissue, the whole hepatic RNA-seq analysis confirmed that only limited and restricted number of genes were changed by alcohol exposure in both models.
As a result, 2 different specific gene signatures of DEGs affected by alcohol exposure in these models (cluster 1 and 2 of DEGs in Fig. 4C,D) support the concept that each models should be considered unique and highly dependent on the qualities of the diet (amount of fat, physical state).
The uniqueness of interaction between alcohol and diet is reflected on our analysis of specific and common DEGs affected by introduction of alcohol in these two models. Not surprisingly, the highly fat enriched LD model is characterized by a specific up-regulation of lipid metabolism gene signature46 as well as glutathione metabolism while MC model has a less discreet effect on genes involved in this process. This close interaction between the diet and alcohol in mouse model is supported by the well-validated observation of relationship between the obesity and ALD progression47.
In spite of limited common pathways affected by alcohol in these two models both of the models validate pnlpα3 as a major and single gene commonly changed by alcohol, fat, and alcohol and fat combination when compared with their respective controls. This is not a surprise considering this gene is well known to increase clinical setting susceptibility not only to non-alcoholic steatohepatitis (NASH)48 but also to ALD49,50. On the other hand, the limited number of genes similarly affected by alcohol in both models raises the question of murine models diversity and mechanistic diversity of ALD in humans based on the environmental factors.
What is especially exciting is that our DEG analysis provides hints for potential biomarkers that may help differentially diagnosis between NASH, ASH, and combination of NASH and ASH. All these entities are more and more intricate in the clinical practice due to an increase of obesity prevalence in the general population, having similar histological features. Oftentimes mild/moderate ASH are difficult to differentiate from NASH with an interobserver variability in recognizing histological features of ASH that is limited and barely reaching 50%51. Based on our analysis, Lcn2 (Lipocalin-2), well known to be affected in murine models using LD model52,53, is actually down-regulated by alcohol in MC model and has potential to make the difference between plain ASH (MC EtOH equivalent) and ASH + NASH (LD EtOH equivalent). Moreover, our DEG analysis, suggested two genes, Elovl6 and Sult2a3, changed by exposure of alcohol in both diets (diet-independent) that can potentially be used to design alcohol specific, diet independent, drug targeting but also potential diet independent biomarkers of alcohol toxicity.
Previous published work showed that alcohol effects on immune system are dependent of the duration and dose of alcohol exposure as well as gender and age of mice, so our present comparative work between these two most common murine chronic models used to study the effect of alcohol has some limitations19,54,55.
First of all, our study is limited to the comparison of only two most common utilized durations of chronic alcohol exposure of 4 weeks for LD diet and 12 weeks of MC model so two different magnitudes of alcohol exposure22,24,25,26,27,28,29. However, it is pretty well known that alcohol effects on specific immune cells and tissue injury are highly dependent of alcohol exposure time56,57,58,59. For example, alcohol decreases NK cell proliferation at 2 weeks while increases splenic NK cell pool at 3 months19.
Secondly, chronic alcohol drinking increases inflammatory transcriptomic profile following LPS stimulation in peripheral blood mononuclear cells in dose dependent manner54. A confounding factor in our study is that the amount of alcohol per day consumed in LD model was higher compared with MC model in spite of more extended time of alcohol exposure in MC mice. For this reason, a comparison of 4 weeks MC model with 4 weeks LD diet potentially decreases the duration of alcohol exposure effect but increases more the differences between the amount of alcohol consumed by the mice in each model.
Third of all, to increase the sensitivity of our analysis we used in our experiments only female mice as is pretty well known that the effect of alcohol is sex dependent in mice and humans55,60,61,62,63,64. Actually, our ongoing current work in the lab is focused on specific cellular immune effects present only in females and not observed in male mice (data not presented, manuscript in preparation).
Last and not least, in order to replicate the most common already published protocols on MC and LD diet, the age of the mice when they start to be exposed to alcohol is pretty young at age of 6 weeks when immune system is still developing. We are very aware of this limitation, so our analysis of the hepatic immune cells is limited only to innate immunity component that is relatively well developed at 6 weeks of age, less age dependent and with a more characterized role in alcoholic liver disease pathogenesis.
In summary our study identifies a very restricted common pathways present in the two most used murine models of chronic alcohol exposure. Furthermore, there are specific cellular and immunological mechanisms highly specific for each model, and some hepatic transcriptome changes present in humans are never seen in murine models in spite of some histological similarities. These data support the concept of the diet specific effect of alcohol effect on the liver; this may be related with the obvious fat content differences but also raises the questions of species-specific mechanisms of alcohol induced organ damage. Whether our observations are the effect of liquid versus solid food (“dynamic action of food state”) or only related with fat-content alone, is at this time matter of additional future investigations. Either way, preclinical testing of targeting alcohol hepatotoxicity effects should consider the model used, followed by a mandatory validation of abnormal pathways present in human targeted organ, and interpreted in the context of highly diet-dependent mechanistic diversity of human and murine alcohol induced pathophysiology.