Discovering Heterogeneous Leukocytes Subsets Associated With Alcoholic Steatohepatitis by scRNAseq Analysis

Abstract

The precise identification of immune cell type responses to alcoholic steatohepatitis (ASH) at the single-cell level remains unresolved. Therefore, in this study, we analyzed heterogeneous immune leukocytes associated with ASH at the single-cell level using high-dimensional single-cell RNA sequencing in alcoholic liver disease (ALD)-induced and healthy control mice. A t-distributed stochastic neighbor embedding plot for dimensionality reduction and 2D visualization was used to visualize heterogeneous immune cell types. Moreover, singleR was used for automated cell annotation to identify the cell types and differentially expressed genes from each cell type and their subsets. We observed a decline in the population of B cells and their subsets, with up and downregulated genes signifying an innate proinflammatory response as an important indication of alcohol-induced liver fibrosis. Additionally, neutrophil deficiency in the alcohol-induced mouse group was associated with ASH. An increase in eosinophils diverts further complications in liver fibrosis, suggesting the functional heterogeneity of granulocyte subsets. Overall, our findings may assist in discovering potential ALD biomarker cell types that are significantly reduced by frequent alcohol exposure and enhance our understanding of the circulating immune leukocytes that lead to alcohol-induced liver fibrosis.

Keywords

alcoholic liver disease / alcoholic steatohepatitis / B cell / circulating immune cells / neutrophils / scRNAseq / tSNEs

References

[1] Y. Zhou, G. Yuan, F. Zhong, and S. He, “Roles of the Complement System in Alcohol-induced Liver Disease,” Clin Mol Hepatol 26 (2020): 677-685.

[2] A. K. Singal, R. Bataller, J. Ahn, P. S. Kamath, and V. H. Shah, “ACG Clinical Guideline: Alcoholic Liver Disease,” American Journal of Gastroenterology 113 (2018): 175-194.

[3] R. Ren, Z. Wang, M. Wu, and H. Wang, “Emerging Roles of SIRT1 in Alcoholic Liver Disease,” Int J Biol Sci 16 (2020): 3174-3183.

[4] J. Mikulak, E. Bruni, F. Oriolo, C. Di Vito, and D. Mavilio, “Hepatic Natural Killer Cells: Organ-Specific Sentinels of Liver Immune Homeostasis and Physiopathology,” Frontiers in Immunology 10 (2019): 946.

[5] L. Li and Z. Zeng, “Live Imaging of Innate and Adaptive Immune Responses in the Liver,” Frontiers in Immunology 11 (2020): 564768.

[6] K. Elvevold, B. Smedsrød, and I. Martinez, “The Liver Sinusoidal Endothelial Cell: A Cell Type of Controversial and Confusing Identity,” Am J Physiol Liver Physiol 294 (2008): G391-400.

[7] H. Peng, E. Wisse, and Z. Tian, “Liver Natural Killer Cells: Subsets and Roles in Liver Immunity,” Cell Mol Immunol 13 (2016): 328-336.

[8] F. Li, C. J. McClain, and W. Feng, “Microbiome Dysbiosis and Alcoholic Liver Disease,” Liver Res 3 (2019): 218-226.

[9] Z. Zhou and W. Zhong, “Targeting the Gut Barrier for the Treatment of Alcoholic Liver Disease,” Liver Res 1 (2017): 197-207.

[10] W.-M. Choi, M.-H. Kim, and W.-I. Jeong, “Functions of Hepatic Non-parenchymal Cells in Alcoholic Liver Disease,” Liver Res 3 (2019): 80-87.

[11] E. Slevin, L. Baiocchi, N. Wu, et al., “Kupffer Cells: Inflammation Pathways and Cell-Cell Interactions in Alcohol-Associated Liver Disease,” American Journal of Pathology 190 (2020): 2185-2193.

[12] G. Szabo, J. Petrasek, and S. Bala, “Innate Immunity and Alcoholic Liver Disease,” Digestive Diseases 30 (2012): 55-60. suppl 1 Suppl. 1.

[13] I. N. Crispe, “The Liver as a Lymphoid Organ,” Annual Review of Immunology 27 (2009): 147-163.

[14] H. Wang, W. Mehal, L. E. Nagy, and Y. Rotman, “Immunological Mechanisms and Therapeutic Targets of Fatty Liver Diseases,” Cell Mol Immunol 18 (2021): 73-91.

[15] S. Mihm, “Danger-Associated Molecular Patterns (DAMPs): Molecular Triggers for Sterile Inflammation in the Liver,” International Journal of Molecular Sciences 19 (2018): 3104.

[16] P. Mandrekar and G. Szabo, “Signalling Pathways in Alcohol-induced Liver Inflammation,” Journal of Hepatology 50 (2009): 1258-1266.

[17] A. Eguchi, R. Yan, S. Q. Pan, et al., “Comprehensive Characterization of Hepatocyte-derived Extracellular Vesicles Identifies Direct miRNA-based Regulation of Hepatic Stellate Cells and DAMP-based Hepatic Macrophage IL-1β and IL-17 Upregulation in Alcoholic hepatitis Mice,” Journal of Molecular Medicine 98 (2020): 1021-1034.

[18] L. Maccioni, B. Gao, S. Leclercq, et al., “Intestinal Permeability, Microbial Translocation, Changes in Duodenal and Fecal Microbiota, and Their Associations With Alcoholic Liver Disease Progression in Humans,” Gut Microbes 12 (2020): 1782157.

[19] B. Seo, K. Jeon, S. Moon, et al., “Roseburia spp. Abundance Associates With Alcohol Consumption in Humans and Its Administration Ameliorates Alcoholic Fatty Liver in Mice,” Cell Host & Microbe 27 (2020): 25-40. e6.

[20] L. Cao, D. Wu, L. Qin, et al., “Single-Cell RNA Transcriptome Profiling of Liver Cells of Short-Term Alcoholic Liver Injury in Mice,” International Journal of Molecular Sciences 24 (2023): 4344.

[21] J. Lotto, S. Drissler, R. Cullum, et al., “Single-Cell Transcriptomics Reveals Early Emergence of Liver Parenchymal and Non-parenchymal,” Cell Lineages Cell 183 (2020): 702-716. e14.

[22] J. Cao, D. R. O'Day, H. A. Pliner, et al., “A human Cell Atlas of Fetal Gene Expression,” Science (80- ) 370 (2020): eaba7721.

[23] X. Zhang, Y. Lan, J. Xu, et al., “CellMarker: A Manually Curated Resource of Cell Markers in human and Mouse,” Nucleic Acids Res 47 (2019): D721-728.

[24] Y. Lv, K. F. So, and J. Xiao, “Liver Regeneration and Alcoholic Liver Disease,” Annals of Translational Medicine 8 (2020): 567.

[25] B. Gao, M. F. Ahmad, L. E. Nagy, and H. Tsukamoto, “Inflammatory Pathways in Alcoholic Steatohepatitis,” Journal of Hepatology 70 (2019): 249-259.

[26] H. Liu, H. Liu, L. Zhu, et al., “Comparative Transcriptome Analyses Provide Potential Insights Into the Molecular Mechanisms of Astaxanthin in the Protection Against Alcoholic Liver Disease in Mice,” Marine Drugs 17 (2019): 181.

[27] J. C. Nawroth, D. B. Petropolis, D. V. Manatakis, et al., “Modeling Alcohol-associated Liver Disease in a human Liver-Chip,” Cell Reports 36 (2021): 109393.

[28] W. Xu, M. Wu, B. Chen, and H. Wang, “Myeloid Cells in Alcoholic Liver Diseases: Mechanism and Prospect,” Frontiers in Immunology 13 (2022): 971346.

[29] J. A. Del Campo, P. Gallego, and L. Grande, “Role of Inflammatory Response in Liver Diseases: Therapeutic Strategies,” World J Hepatol 10 (2018): 1-7.

[30] E. M. Brunt, D. E. Kleiner, D. H. Carpenter, et al., “NAFLD: Reporting Histologic Findings in Clinical Practice,” Hepatology 73 (2021): 2028-2038.

[31] Z. Galvin, A. McDonough, J. Ryan, and S. Stewart, “Blood Alanine Aminotransferase Levels >1,000 IU/L—causes and Outcomes,” Clin Med (Northfield Il) 15 (2015): 244-247.

[32] Y. Liu, L.-S. Cheng, S. Wu, et al., “IL-10-producing Regulatory B-cells Suppressed Effector T-cells but Enhanced Regulatory T-cells in Chronic HBV Infection,” Clinical Science (London, England: 1979) 130 (2016): 907-919.

[33] A. Zwolak, A. Surdacka, and J. Daniluk, “Bcl-2 and Fas Expression in Peripheral Blood Leukocytes of Patients With Alcoholic and Autoimmune Liver Disorders,” Human & Experimental Toxicology 35 (2015): 799-807.

[34] Z.-X. Li, Q.-N. Zhu, H.-B. Zhang, Y. Hu, G. Wang, and Y.-S. Zhu, “MALAT1: A Potential Biomarker in Cancer,” Cancer Manag Res 10 (2018): 6757-668.

[35] E. S. Lee, E. J. Wolf, S. S. J. Ihn, H. W. Smith, A. Emili, and A. F. Palazzo, “TPR Is Required for the Efficient Nuclear Export of mRNAs and lncRNAs From Short and Intron-poor Genes,” Nucleic Acids Res 48 (2020): 11645-1163.

[36] S. S. Ng, F. De Labastida Rivera, J. Yan, et al., “The NK Cell Granule Protein NKG7 Regulates Cytotoxic Granule Exocytosis and Inflammation,” Nature Immunology 21 (2020): 1205-1218.

[37] L. Li, Z. Pan, and X. Yang, “Key Genes and co-expression Network Analysis in the Livers of Type 2 Diabetes Patients,” J Diabetes Investig 10 (2019): 951-962.

[38] V. Aksenova, H. N. Lee, A. Smith, et al., “Distinct Basket Nucleoporins Roles in Nuclear Pore Function and Gene Expression: Tpr Is an Integral Component of the TREX-2 mRNA Export Pathway,” BioRxiv (2019), https://doi.org/10.1101/685263.

[39] L. Bao, A. F. Odell, S. L. Stephen, S. B. Wheatcroft, J. H. Walker, and S. Ponnambalam, “The S100A6 Calcium-binding Protein Regulates Endothelial Cell-cycle Progression and Senescence,” Febs Journal 279 (2012): 4576-4588.

[40] A. Alijagic, D. Gaglio, E. Napodano, et al., “Titanium Dioxide Nanoparticles Temporarily Influence the Sea Urchin Immunological state Suppressing Inflammatory-relate Gene Transcription and Boosting Antioxidant Metabolic Activity,” Journal of Hazardous Materials 384 (2020): 121389.

[41] E. Ghosn, K. Dorshkind, L. A. Herzenberg, et al., “B1 B Cell Progenitors,” Science (80- ) 364 (2019): 248.

[42] T. Vogl, A. L. Gharibyan, and L. A. Morozova-Roche, “Pro-Inflammatory S100A8 and S100A9 Proteins: Self-Assembly Into Multifunctional Native and Amyloid Complexes,” International Journal of Molecular Sciences 13 (2012): 2893-2917.

[43] R. Acin-Perez, E. Salazar, S. Brosel, H. Yang, E. A. Schon, and G. Manfredi, “Modulation of Mitochondrial Protein Phosphorylation by Soluble Adenylyl Cyclase Ameliorates Cytochrome Oxidase Defects,” EMBO Molecular Medicine 1 (2009): 392-406.

[44] H. Xue, R. L. McCauley, and W. Zhang, “Elevated Interleukin-6 Expression in Keloid Fibroblasts,” Journal of Surgical Research 89 (2000): 74-77.

[45] X. Shi, A. L. DeLucia, J. Bao, and P. Zhang, “Alcohol Abuse and Disorder of Granulopoiesis,” Pharmacology & Therapeutics 198 (2019): 206-219.

[46] L. N. C. Moscinski and B. Hill, “Molecular Cloning of a Novel Myeloid Granule Protein,” Journal of Cellular Biochemistry 59 (1995): 431-442.

[47] J. H. White, “Vitamin D as an Inducer of Cathelicidin Antimicrobial Peptide Expression: Past, Present and Future,” Journal of Steroid Biochemistry and Molecular Biology 121 (2010): 234-238.

[48] A. Zare, D. Henry, G. Chua, P. Gordon, and H. R. Habibi, “Differential Hepatic Gene Expression Profile of Male Fathead Minnows Exposed to Daily Varying Dose of Environmental Contaminants Individually and in Mixture,” Front Endocrinol (Lausanne) 9 (2018): 749.

[49] D. Keppler, “Towards Novel Anti-cancer Strategies Based on Cystatin Function,” Cancer Letters 235 (2006): 159-176.

[50] G. A. Ramirez, M.-R. Yacoub, M. Ripa, et al., “Eosinophils From Physiology to Disease: A Comprehensive Review,” BioMed Research International 2018 (2018): 9095275.

[51] I. Novita Sari, Y.-G. Yang, L. T. Hanh Phi, et al., “Interferon-induced Transmembrane Protein 1 (IFITM1) Is Required for the Progression of Colorectal Cancer,” Oncotarget 7 (2016): 86039-89050.

[52] S. Yona, K.-W. Kim, Y. Wolf, et al., “Fate Mapping Reveals Origins and Dynamics of Monocytes and Tissue Macrophages Under Homeostasis,” Immunity 38 (2013): 79-91.

[53] H. J. Wang, B. Gao, S. Zakhari, and L. E. Nagy, “Inflammation in Alcoholic Liver Disease,” Annual Review of Nutrition 32 (2012): 343-368.

[54] Y. Choi, H. Choi, B. K. Yoon, et al., “Serpina3c Regulates Adipogenesis by Modulating Insulin Growth Factor 1 and Integrin Signaling,” Iscience 23 (2020): 100961.

[55] C. Shengwen, L. Qian, Z. Lini, et al., “The Role of REC8 in the Innate Immune Response to Viral Infection,” Journal of Virology 96 (2022): e02175-21.

[56] Z. Wang, R. Huang, H. Wang, et al., “Prognostic and Immunological Role of PPP1R14A as a Pan-Cancer Analysis Candidate,” Frontiers in Genetics 13 (2022): 842975.

[57] W. Udomsinprasert, N. Angkathunyakul, J. Jittikoon, et al., “Cartilage Oligomeric Matrix Protein as a Marker of Progressive Liver Fibrosis in Biliary Atresia,” Scientific Reports 11 (2021): 16695.

[58] X. Wang, Y. Xiao, S. Li, Z. Yan, and G. Luo, “CORO6 Promotes Cell Growth and Invasion of Clear Cell Renal Cell Carcinoma via Activation of WNT Signaling,” Frontiers in Cell and Developmental Biology 9 (2021): 647301.

[59] Q. Gao, X. Chen, H. Duan, et al., “FXYD6: A Novel Therapeutic Target Toward Hepatocellular Carcinoma,” Protein Cell 5 (2014): 532-543.

[60] H. Zhang, Q. Kong, J. Wang, Y. Jiang, and H. Hua, “Complex Roles of cAMP-PKA-CREB Signaling in Cancer,” Exp Hematol Oncol 9 (2020): 32.

[61] H. H. Nam, D. W. Jun, K. Jang, et al., “Granulocyte Colony Stimulating Factor Treatment in Non-alcoholic Fatty Liver Disease: Beyond Marrow Cell Mobilization,” Oncotarget 8 (2017): 97965-97976.

[62] H.-N. Xuan Yuan, H. S. Kim, G. R. Park, et al., “Adenosine Triphosphate-binding Pocket Inhibitor for Mixed Lineage Kinase Domain-Like Protein Attenuated Alcoholic Liver Disease via Necroptosis-independent Pathway,” World Journal of Gastroenterology 31 (2025): 96782.

[63] V. Patra, N. Woltsche, U. Cerpes, et al., “Persistent Neutrophil Infiltration and Unique Ocular Surface Microbiome Typify Dupilumab-Associated Conjunctivitis in Patients With Atopic Dermatitis,” Ophthalmology Science 4, no. 1 (2024): 100340.

[64] F. Veglia, A. Hashimoto, H. Dweep, et al., “Analysis of Classical Neutrophils and Polymorphonuclear Myeloid-Derived Suppressor Cells in Cancer Patients and Tumor-Bearing Mice,” Journal of Experimental Medicine 218, no. 4 (2021).

[65] Z. Bagheri-Hosseinabadi, M. Abbasi, M. Kahnooji, Z. Ghorbani, and M. Abbasifard, “The Prognostic Value of S100A Calcium Binding Protein Family Members in Predicting Severe Forms of COVID-19,” Inflammation Research 71, no. 3 (2022): 369-376.

[66] S. A. Ragland and A. K. Criss, “From Bacterial Killing to Immune Modulation: Recent Insights Into the Functions of Lysozyme,” PLOS Pathogens 13, no. 9 (2017): e1006512.

[67] T. Girbl, T. Lenn, L. Perez, et al., “Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis,” Immunity 49, no. 6 (2018): 1062-1076.e6.

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