ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. PULMONARY INFLAMMATION & INJURY IN A MOUSE MODEL OF NON-ALCOHOLIC STEATOHEPATITIS TANVI BANOTA, ALEXA MURRAY, LAURA E. ARMSTRONG, BO KONG, GRACE L. GUO, ANDREW J. GOW, DEBRA L. LASKIN (FACULTY ADVISOR) โœต ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is a chronic liver condition that affects millions of individuals in the United States, of which approximately twenty percent of cases progress to non-alcoholic steato- hepatitis (NASH). NASH is characterized by macro- vascular steatosis and persistent inflammation in the liver, which can lead to fibrosis. Evidence suggests potential effects of NAFLD and NASH on the devel- opment of pulmonary pathologies, but the interac- tion between the liver and the lung is not well under- stood. In this study, we assessed the impact of NASH development on lung inflammation and fibrosis over time. Male C57BL/6J mice were fed control (10% kCal) or high-fat (HFD) (60% kCal) diets. Liver tissue, lung tissue, and bronchoalveolar lavage (BAL) fluid were collected after 1, 3, and 6 months of feeding. Histopathologic evaluation of livers from HFD-fed mice at 6 months confirmed the development of NASH. In the lung, we observed histopathologic al- terations, including inflammatory cell infiltration, li- pid-laden macrophages, septal damage, and epi- thelial thickening at 6 months. Gene expression anal- ysis of whole lung tissue revealed changes in genes related to inflammation (IL-1B), fibrosis (CTGF), and lipid metabolism (ApoA1). These results characterize an association of pulmonary complications during simple steatosis to NASH transition, suggesting lung-liver crosstalk. 1 INTRODUCTION Non-alcoholic fatty liver disease (NAFLD) is a chronic liver condition that is estimated to affect upwards of thirty percent of individuals in the United States, with even more at risk due to the rising obe- sity epidemic.[3,5,19] NAFLD is characterized by the ac- cumulation of fat in the liver, known as steatosis. It is estimated that twenty percent of patients diagnosed with NAFLD progress to non-alcoholic steatohepati- tis (NASH).[21] NASH is a severe, chronic liver disease characterized by persistent inflammation and im- mune cell infiltration. NASH may also progress to fi- brosis and cirrhosis of the liverโ€”this irreversible scar- ring of tissue can further lead to uncontrolled cell growth, cancer, and death. NASH is becoming the leading indication for liver transplantation in both the U.S. and worldwide.[2,14,16] Both NAFLD and NASH are associated with systemic effects that manifest in pathologies across the body, including cardiovascular disease, meta- bolic syndrome, and chronic kidney conditions.[1] Emerging evidence also suggests that NAFLD and NASH may impact the development of pathologies in the lung. Several longitudinal observational stud- ies have detailed an association between NAFLD and decreased measurements of lung function (e.g. forced expiratory volume and vital capacity).[7,11,12,15] In addition, patients with chronic obstructive pulmo- nary disease (COPD) showed increased incidence of both NAFLD and NASH.[20] Although there is rising clinical evidence of a relationship between NASH and reduced pulmonary function, the interplay be- tween the liver and the lung remains largely unex- plored. A central aspect of NAFLD and NASH that may contribute to lung injury is inflammation. Key players in this response are macrophages, phago- cytic cells of the innate immune system. In the liver, macrophages are known to take up surrounding fat through endocytosis. This causes the macrophages to become activated and release pro-inflammatory mediators such as cytokines, small proteins im- portant in inflammatory cell signaling.[14] These me- diators enter the bloodstream and can exert effects in other tissues of the body through systemic circu- ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III lation, leading to observed comorbidities.[1,9,14] We hypothesize that these inflammatory mediators ac- cumulate in the lung, causing pulmonary injury, in- flammation, and the disruption of key signaling path- ways by dysregulating genes related to lipid metab- olism and inflammation, including Il-1B, Ctgf, Lxr, ApoA1, and Abca1. The present study was designed to test this hypothesis and characterize the develop- ment of lung injury in a high fat diet mouse model of NASH. The results of our study provide data on lung- liver crosstalk, which may be useful for the develop- ment of new approaches for clinical management of pulmonary pathology related to NASH. 2 METHODOLOGY ANIMALS & TREATMENTS Wild type male C57BL/6J mice (6-8 weeks, ๐‘›๐‘› = 5 โˆ’ 9/group) were fed control (10% kCal) or a high-fat diet (HFD) (60% kCal) for 1, 3, and 6 months. Food consumption was monitored weekly. Animal protocols were approved by Rutgers University IACUC. HISTOLOGICAL ANALYSIS Liver and lung tissue were collected, fixed in 10% for- malin or inflated and fixed in 4% paraformaldehyde, respectively, and cut into 5-ฮผm sections and stained with hematoxylin & eosin (H&E). Lung sections were evaluated for characteristics of lung injury and in- flammation, including inflammatory cell infiltration, septal damage, and epithelial thickening. Liver sec- tions were examined for the histopathological char- acteristics of NAFLD (e.g. fat accumulation) and NASH (e.g. fat accumulation and inflammatory cell infiltration) based on established criteria.[2,16] BRONCHOALVEOLAR LAVAGE (BAL) CELL AND PROTEIN MEASUREMENT BAL fluid was collected by slowly instilling and withdraw- ing 1 mL of ice-cold (4ยฐC) PBS into the lungs of mice through a cannula in the trachea (FIGURE 1). This fluid was centri- fuged at 300xg for 8 minutes. Cell pellets were resuspended in 1 mL of PBS. Viable cells (10 ฮผl) were counted on a hemocytometer using trypan blue dye exclusion. Cytospins were prepared by centrifugation of 104 cells BAL fluid onto microscope slides using a Shan- don cytospin (Thermo Scientific). Cells were fixed in methanol and stained with Giemsa to visualize BAL cell populations. Total protein content in cell free BAL was quantified using a BCA protein kit (Pierce Biotechnologies Inc.) with bovine serum albumin as the standard. All samples were assayed in triplicate at 562 nm using a spectrophotometer. MRNA ISOLATION AND RT-QPCR ANALYSIS Total RNA was extracted from lung tissue using TRI- zol๏›› Reagent and bead TissueLyser LT (Qiagen). cDNA was generated using High Capacity cDNA Re- verse Transcription kit (Applied Biosystems). Real- time quantitative PCR (RT-qPCR) was performed on a QuantStudio 6 system using commercially availa- ble Power SYBRยฎ Green gene expression assays (Applied Biosystems). Data were normalized to ฮฒ-ac- tin and presented as fold change relative to 1 month CTRL mice. Fold changes in gene expression were calculated using โˆ†โˆ†๐ถ๐ถ๐ถ๐ถ method where ๐ถ๐ถ๐ถ๐ถ(๐ถ๐ถ๐‘ก๐‘ก๐‘ก๐‘ก๐‘ก๐‘ก๐‘ก๐‘ก๐ถ๐ถ ๐‘ก๐‘ก๐‘ก๐‘ก๐‘›๐‘›๐‘ก๐‘ก) โ€“ ๐ถ๐ถ๐ถ๐ถ(๐›ฝ๐›ฝ โˆ’ ๐‘ก๐‘ก๐‘Ž๐‘Ž๐ถ๐ถ) = โˆ†๐ถ๐ถ๐ถ๐ถ; โˆ†๐ถ๐ถ๐ถ๐ถ โ€“ ๐‘ก๐‘ก๐‘Ž๐‘Ž๐‘ก๐‘ก๐‘ก๐‘ก๐‘ก๐‘ก๐‘ก๐‘ก๐‘ก๐‘ก โˆ†๐ถ๐ถ๐ถ๐ถ(1 ๐‘€๐‘€๐‘€๐‘€๐‘›๐‘›๐ถ๐ถโ„Ž ๐ถ๐ถ๐ถ๐ถ๐ถ๐ถ๐ถ๐ถ) = โˆ†โˆ†๐ถ๐ถ๐ถ๐ถ; and 2 โˆ’ โˆ†โˆ†๐ถ๐ถ๐ถ๐ถ = ๐‘“๐‘“๐‘€๐‘€๐‘“๐‘“๐‘“๐‘“ ๐‘Ž๐‘Žโ„Ž๐‘ก๐‘ก๐‘›๐‘›๐‘ก๐‘ก๐‘ก๐‘ก. STATISTICAL ANALYSIS Data are presented as mean + SE and were analyzed using 2-way ANOVA and Sidakโ€™s multiple compari- sons test. A p-value โ‰ค0.05 was considered statisti- cally significant. 3 RESULTS HIGH-FAT DIET INDUCES NASH AND CAUSES HISTOPATHOLOGICAL CHANGES IN THE LUNG To confirm the development of NASH, we assessed histopathological changes in the liver. Livers from mice fed a HFD for 1 and 3 months exhibited steato- sis, or the accumulation of lipid droplets in the liver, but no inflammatory cell infiltration, indicating NAFLD (data not shown). The most prominent changes in the liver were observed 6 months follow- ing consumption of a HFD; these included more se- vere steatosis and infiltration of inflammatory cells FIGURE 1 ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III (FIGURE 2A). Significant increases in body weights, in- creases in total serum cholesterol, and decreased glucose tolerance in HFD-fed mice confirmed this was metabolic syndrome-related NASH (data not shown). We next assessed alterations in lung histol- ogy. After 6 months, inflammatory cell infiltration, septal damage, and epithelial thickening were ob- served in HFD-fed mice relative to mice fed the con- trol diet (FIGURE 2B). The accumulation of large macro- phages in the lung that appeared lipid-laden was also noted in the histology. Further examination of cell cytospins also revealed the presence of large, vacuolated, and potentially lipid-laden macro- phages (FIGURE 2B inserts). We further investigated lung injury and inflammation by quantifying levels of BAL protein and cells, respectively (FIGURE 2C). Alt- FIGURE 2: Representative images of H&E stained sections of liver (PANEL A) and lung (PANEL B) from mice fed control (CTRL) or high fat diets (HFD) for 6 months. PANEL A, left arrow indicates steatosis, right arrow and inset indicates inflammation. PANEL B, top arrow indicates inflammatory cell infiltration and bottom arrow indicates epithelial thickening. PANEL B insets highlight rep- resentative macrophages from Giemsa-stained cytopsins, including macrophages with a large, lipid-laden appearance in HFD-fed mice. Original magnification (A) 4x or (B) 20x, inserts 40x. (C) BAL collected from mice 1, 3, and 6 months after a control (CTRL) or high fat diet (HFD) was assessed for protein and cell content. Bars, mean + SE (n=5-10). *Significantly different from CTRL fed mice. #Significantly different from 1 month. aSignificantly different from 3 month. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III hough increased lung injury was noted in HFD-fed mice at 6 months as measured by BAL protein, this may be due to a reduction in BAL protein in control mice at this time. Surprisingly, a time related in- crease in lung inflammation was observed in control mice at 3 and 6 months. Cell counts were unaffected by administration of the HFD. HIGH FAT DIET DISRUPTS EXPRESSION OF INFLAMMATORY AND LIPID METABOLISM RELATED GENES To better understand degrees of inflammatory changes in the lung following a HFD, we analyzed expression of inflammatory and lipid-related genes. Expression of interleukin 1 beta (Il-1b), a key early re- sponse proinflammatory gene, was significantly in- creased in mice fed a HFD when compared to con- FIGURE 3: Lung tissue collected 1, 3, and 6 months after control (CTRL) or high fat diet (HFD) from mice were analyzed for gene expression by RT-qPCR. Data were normalized relative to รŸ-actin and presented as fold change relative to 1 Month CTRL fed mice. Bars, mean + SE (n=3-7). *Significantly different from CTRL fed mice. #Significantly different from 1 month. aSignificantly different from 3 month. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III trol mice at 3 months. Il-1b expression returned to control levels by 6 months. The expression of con- nective tissue growth factor (Ctgf), a gene indicative of fibrosis and tissue remodeling, was significantly decreased 1 month following HFD-feeding. Interest- ingly, control mice displayed a significant decrease in Ctgf at 3 and 6 months when compared to 1 month. Similarly, expression of apolipoprotein A1 (Apoa1), a lipid chaperone, was decreased 1 month following HFD when compared to control-fed mice. ApoA1 expression was similarly reduced in the 6- month control-fed mice when compared to 1-month control mice. There were no changes in expression of liver X receptor (Lxr), a nuclear receptor involved in lipid homeostasis, and its target ATP-binding cas- sette transporter 1 (Abca1), a lipid transporter. 4 DISCUSSION The inflammatory and fibrogenic effects of NASH in the liver have been well characterized.[2,14] Emerging clinical evidence suggests that NAFLD and NASH are associated with pulmonary injury, but crosstalk between the lung and the liver in NASH has not been investigated.[7,11,12,15] In these studies, we used a mouse model of HFD to induce NASH and investigate associated injury and inflammation in the lung. We found that NASH was associated with his- topathological alterations in lung tissue 6 months post HFD feeding; moreover, expression of genes related to inflammation and lipid metabolism was dysregulated throughout NAFLD development, in- cluding the progression to NASH. These results pro- vide insights into the interplay between liver and lung inflammation and highlight potential inflamma- tory pathways for crosstalk between the tissues. Based on established criteria, we confirmed the development of NASH in mice fed a HFD as we observed increased steatosis and inflammatory cell infiltration at 6 months.[2,16] This was correlated with pulmonary histopathological changes in the HFD- fed mice at this time. Further assessment by a pathologist will be completed to confirm these his- topathological changes. Although there was no evi- dence of pulmonary fibrosis in these animals, we noted the appearance of lipid-laden macrophages in the lung. These cells have been shown to be asso- ciated with fibrosis in other disease states, and may contribute to the development of lung fibrosis at later time points in NASH.[18] Interestingly, histo- pathological changes in the lung were not reflected by increases in BAL protein or cell counts, which are markers of pulmonary alveolar epithelial damage and leaky vasculature, or infiltration of immune cells into the lung in response to injury.[17] It may be that NASH is not associated with epithelial barrier dys- function and that pathologic alterations involve other mechanisms of injury. For example, it is possi- ble that resident macrophages present in the lung are activated following HFD and that they drive lung inflammation. Further studies are needed to explore this possibility. We also noted a decrease in BAL pro- tein content at 6 months and increases in BAL cells in control mice; this may be indicative of age-related changes in tissue structure or vasculature, or in basal inflammatory activity.[4] HFD-fed mice seem to also mimic this age-related trend of increasing inflamma- tion, although not significantly. We speculated that histopathological changes in the lung of animals fed a HFD might be driven by differential expression of genes related to inflammatory proteins and cytokines. In this context, our gene expression analyses revealed increases in Il-1b at 3 months in HFD-fed mice, which suggests that during the development of NASH, the lung re- sponds to hepatic inflammation by upregulating in- flammatory gene expression. IL-1ฮฒ is an early re- sponse cytokine known to promote inflammation; thus, increases in inflammatory cells in the lung of mice fed a HFD may be mediated in part by this cy- tokine. We also observed early downregulation of Apoa1 in HFD-fed mice at 1 month. ApoA1 is a lipid chaperone that promotes the efflux of cholesterol; it has been shown to have anti-inflammatory and anti- fibrotic effects in the lung.[6,8] The observed decrease in ApoA1 may further exacerbate inflammation in the lung in response to a HFD. Although we did not observe lung fibrosis during the histopathological analysis, we assessed changes in CTGF gene expres- sion as a measure of fibrotic extracellular matrix tis- sue remodeling.[10,13] Ctgf expression was decreased in HFD-fed mice at 1 month, suggesting that a HFD may suppress fibrotic mechanisms in the lung early ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III in the process of NASH development. This might be a compensatory response to prevent fibrosis in- duced by other growth factors generated in the lung in response to the HFD. A similar decrease in Ctgf expression at 3 and 6 months in the control mice in- dicates that the HFD may be mimicking age-related effects in mice as early as 1 month. While these data provide preliminary char- acterization of pulmonary changes in a mouse model of NASH, experiments to confirm the pres- ence of lipid-laden macrophages by staining for li- pids as well as an assessment of histopathological changes by a pathologist will be conducted. Future studies will also be performed to further elucidate mechanisms of high fat diet-associated effects on the lung, including assessment of systemic markers of lipid dysregulation, liver inflammation and dys- function, and other inflammatory signaling pathways in the lung. Developing our understanding of the in- terplay between the lung and the liver can help iden- tify the mechanisms by which disease can influence distant pathologies, and how inflammation in partic- ular can be controlled to limit pathological comor- bidities in patients. 5 CONCLUSION Overall, these data demonstrate that HFD- induced NASH leads to pulmonary histopathologi- cal changes. Moreover, these changes may be driven by the dysregulation of key mediators in- volved in inflammation and lipid metabolism. This analysis of lung-liver crosstalk in NASH highlights po- tential for the clinical management of pulmonary complications associated with NASHโˆŽ 6 ACKNOWLEDGEMENTS I would like to acknowledge everyone in Dr. Debra Laskinโ€™s lab for their guidance and support, espe- cially Dr. Alexa Murray and Dr. Debra Laskin. I would also like to acknowledge Dr. Bo Kong for his exper- tise and guidance with the animals used in this pro- ject, Dr. Laura Armstrong for providing liver images, and Dr. Grace Guo for her continued scientific guid- ance. This research was supported by NIH Grants ES029258, ES005022, and ES004738. 7 REFERENCES [1] Armstrong, M.J., Adams, L.A., Canbay, A., & Syn, W.K. (2014). Extrahepatic complications of nonalcoholic fatty liver disease. Hepatology, 59(3), 1174โ€“1197. [2] Benedict, M., & Zhang, X. (2017). Non-alcoholic fatty liver dis- ease: An expanded review. World Journal of Hepatology, 9(16), 715โ€“732. [3] Fabbrini, E., Sullivan, S., & Klein, S. (2010). Obesity and Non- alcoholic Fatty Liver Disease: Biochemical, Metabolic and Clinical Implications. Hepatology (Baltimore, Md.), 51(2), 679โ€“689. [4] Franceschi, C., Garagnani, P., Parini, P., Giuliani, C., & San- toro, A. (2018). Inflammaging: A new immune-metabolic viewpoint for age-related diseases. Nature Reviews. 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[13] Ponticos, M., Holmes, A.M., Shi-wen, X., Leoni, P., Khan, K., Rajkumar, V.S., Hoyles, R.K., Bou-Gharios, G., Black, C.M., Denton, C.P., Abraham, D.J., Leask, A., & Lindahl, G.E. (2009). Pivotal role of connective tissue growth factor in lung fibrosis: MAPK-dependent transcriptional activation of type I collagen. Arthritis and Rheumatism, 60(7), 2142โ€“2155. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE III [14] Schuster, S., Cabrera, D., Arrese, M., & Feldstein, A.E. (2018). Triggering and resolution of inflammation in NASH. Nature Reviews Gastroenterology & Hepatology, 15(6), 349โ€“364. [15] Song, J.U., Jang, Y., Lim, S.Y., Ryu, S., Song, W.J., Byrne, C.D., & Sung, K.C. (2019). Decreased lung function is associated with risk of developing non-alcoholic fatty liver disease: A longitudinal cohort study. PLoS ONE, 14(1). [16] Spengler, E.K., & Loomba, R. (2015). Recommendations for Diagnosis, Referral for Liver Biopsy, and Treatment of NAFLD and NASH. Mayo Clinic Proceedings, 90(9), 1233โ€“1246. [17] Sunil, V.R., Patel, K.J., Shen, J., Reimer, D., Gow, A.J., Laskin, J.D., & Laskin, D.L. (2011). Functional and inflammatory alter- ations in the lung following exposure of rats to nitrogen mus- tard. Toxicology and Applied Pharmacology, 250(1), 10โ€“18. [18] Venosa, A., Smith, L.C., Murray, A., Banota, T., Gow, A.J., Las- kin, J.D., & Laskin, D.L. (2019). Regulation of Macrophage Foam Cell Formation During Nitrogen Mustard (NM)-In- duced Pulmonary Fibrosis by Lung Lipids. Toxicological Sci- ences, 172(2), 344โ€“358. [19] Vernon, G., Baranova, A., & Younossi, Z.M. (2011). Systematic review: The epidemiology and natural history of non-alco- holic fatty liver disease and non-alcoholic steatohepatitis in adults. Alimentary Pharmacology & Therapeutics, 34(3), 274โ€“ 285. [20] Viglino, D., Plazanet, A., Bailly, S., Benmerad, M., Jullian-De- sayes, I., Tamisier, R., Leroy, V., Zarski, J.P., Maignan, M., Joyeux-Faure, M., & Pรฉpin, J.L. (2018). Impact of Non-alco- holic Fatty Liver Disease on long-term cardiovascular events and death in Chronic Obstructive Pulmonary Disease. Scien- tific Reports, 8. [21] Wong, R.J., Aguilar, M., Cheung, R., Perumpail, R.B., Harri- son, S.A., Younossi, Z.M., & Ahmed, A. (2015). Nonalcoholic Steatohepatitis Is the Second Leading Etiology of Liver Dis- ease Among Adults Awaiting Liver Transplantation in the United States. Gastroenterology, 148(3), 547โ€“555. Tanvi Banota is a senior in the Honors College at Rutgers University majoring in Cell Biology and Neuroscience and minoring in Linguistics. She has been conducting research in the Laskin Lab since high school when she was first matched with the lab through the Liberty Science Center Part- ners in Science Program. Her research is on the mechanisms of inflammation following toxicant- induced pulmonary injury. Tanvi plans to pursue an MD/PhD after Rutgers and hopes to have a career running her own lab, seeing patients, and mentoring students. Outside of academics and research, Tanvi is an epee fencer on the Rutgers Fencing Team, is highly involved with the Aresty Research Center, and enjoys watching sports, spending time with her friends, and playing and lis- tening to music. Tanvi was also a part of the founding team for the Aresty RURJ in 2019 and has served as a peer reviewer, senior peer reviewer, and editor for the journal. Sheโ€™s excited to see where the journal will go next and how it will continue to uplift and represent the undergraduate research community at Rutgers! For any questions, please contact Tanvi at: TANVI.BANOTA@RUTGERS.EDU