bioRxiv preprint doi: https://doi.org/10.1101/600676; this version posted April 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 1 The Role of CD147 in Leukocyte Aggregation in Liver Injury 2 3 Christine Yee1,2,*, Nathan Main2,*, Alexandra Terry1,2,*, Igor Stevanovski2, 4 Annette Maczurek1, Alison J. Morgan1, Sarah Calabro1, Alison J. Potter1, Tina 5 L. Iemma1, David G. Bowen1,3, Golo Ahlenstiel4, Fiona J. Warner1, Geoffrey W. 6 McCaughan1,3, Susan V. McLennan5 and Nicholas A. Shackel1,2,3,6 7 8 1Centenary Research Institute, University of Sydney, New South Wales, 9 Australia. 10 2Gatsroneterology and Liver Laboratory Ingham Institute, Liverpool, NSW, 11 Australia. 12 3A.W. Morrow Gastroenterology and Liver Centre, Royal Prince Alfred Hospital, 13 Camperdown, NSW, Australia. 14 4Western Sydney school of Medicine, Blacktown Hospital, Blacktown, NSW, 15 Australia. 16 5Department of Endocrinology, Department of Medicine and Bosch Institute, 17 Royal Prince Alfred Hospital, University of Sydney, Australia. 18 6Liverpool Hospital, Liverpool, NSW, Australia. 19 20 * Equal contribution 21 22 Corresponding Author: Prof. Nicholas A. Shackel 23 Ingham Institute, 24 1 Campbell St 25 Liverpool, NSW, 2170 26 Australia 27 Email: [email protected] 28 29 30 31 32 33 34 bioRxiv preprint doi: https://doi.org/10.1101/600676; this version posted April 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 2 35 Abstract (Words 247) 36 37 Background: Chronic inflammation is the driver of liver injury resulting in 38 progressive fibrosis and eventual cirrhosis. The consequences include both 39 liver failure and liver cancer. We have previously described increased 40 expression of the highly multifunctional glycoprotein CD147 in liver injury. This 41 work describes a novel role of CD147 in liver inflammation and the importance 42 of leukocyte aggregates in determining the extent of liver injury. 43 44 Methods: Non-diseased, progressive injury and cirrhotic liver from humans and 45 mice were examined using mAb targeting CD147. Inflammatory cell subsets 46 were assessed by multicolor flow cytometry. 47 48 Results: In liver injury, we observe abundant intrahepatic leukocyte clusters 49 defined as ≥5 adjacent CD45+ cells which we have labelled “leukocyte 50 aggregates”. We have shown that these leukocyte aggregates are significant 51 in determining the extent of liver injury. If CD147 is blocked in vivo, these 52 leukocyte aggregates diminish in size and number together with a marked 53 significant reduction in liver injury including fibrosis. This accompanied by no 54 change in overall intrahepatic leukocyte numbers. Further, blocking 55 aggregation formation occurs prior to an appreciable increase in inflammatory 56 markers or fibrosis. Additionally, there were no observed, “off-target” or 57 unpredicted effects in targeting CD147. 58 59 Conclusion: CD147 mediates leukocyte aggregation which is associated with 60 the development of liver injury. This is not a secondary effect, but a cause of 61 injury as aggregate formation proceeds other markers of injury. Leukocyte 62 aggregation has been previously described in inflammation dating back over 63 many decades but till now been shown to determine the extent of injury. 64 65 66 67 68 bioRxiv preprint doi: https://doi.org/10.1101/600676; this version posted April 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 3 69 Introduction 70 71 The classical hallmark of liver injury is the deposition of abnormal/fibrotic 72 extracellular matrix (ECM) and the eventual development of cirrhosis which is 73 mediated by the activated hepatic stellate cell (HSC)(1). Chronic inflammation 74 drives ongoing HSC activation and fibrosis(1). Chronic liver inflammation can be 75 regarded as commencing with an initial innate immune response to an ongoing 76 insult that develops into chronic injury with both sustained innate and adaptive 77 immune components(2-6). T-cell responses are clearly pivotal to the 78 development of chronic immune-mediated hepatic injury and it has now been 79 shown that B-cells are essential for the development of intrahepatic fibrosis 80 leading to cirrhosis(7-11). 81 Importantly, we have previously identified a number of novel pathways 82 of liver injury(12-14). Arising from these studies we have shown that hepatocytes 83 remodel extracellular matrix in liver injury via production of active matrix- 84 metalloproteinases(15). 85 CD147 is an abundant 269aa type 1 integral glycosylated multifunctional 86 membrane protein with differing cellular functions on differing cell sub- 87 populations. The concept of multifunctional proteins is well established(16-20) 88 with at least 3% of all proteins in the human protein interaction network being 89 ‘extreme multifunctional’ proteins(20). CD147 is expressed within a wide range 90 of tissues, including endothelium and epithelium(21-23). CD147 is thought to act 91 via MAPK p38, ERK-1, -2, PI3K and NF-ΚB signaling pathways(24-27). The 92 regulation of CD147 expression is largely uncharacterised. 93 A striking and consistent function attributed to CD147 is its ability to 94 regulate leukocyte chemotaxis. Cyclophilins (CyP)-A and B are the two CD147 95 ligands known to mediate chemotaxis(21, 28-30). CD147 has been shown to be 96 important for leukocyte recruitment in rheumatoid arthritis, multiple sclerosis 97 and inflammatory lung disease, with mAb αCD147 interventions leading to 98 reduced neutrophil, T-cell and monocytes/macrophage infiltration(11, 28, 31-37). 99 CD147 is mostly found in membrane protein complexes(29, 30, 38-40) and 100 many of these binding partners are matrix components or inflammatory 101 mediators that are dramatically increased with injury (i.e. hyaluronan(41), 102 intercellular adhesion molecule (ICAM)-1(38, 42, 43), lymphocyte function- bioRxiv preprint doi: https://doi.org/10.1101/600676; this version posted April 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 4 103 associated antigen (LFA)-1(44-46) and CD43(47, 48)). Additionally, CD147 is also 104 found in large complicated multiple protein “super-complexes” on the cell 105 surface containing many binding partners(39). Studies have shown that the 106 domain of CD147 that mediates cell migration is distinct from the domain 107 mediating MMP induction (within extracellular loop I)(49, 50). Therefore, it is well 108 established that CD147 is a multifunctional protein(16-20) with distinct binding 109 partners and structural domains determining its varied functions at specific 110 cellular locations. 111 Leukocyte aggregations of either lymphoid or myeloid origin are thought 112 to exacerbate inflammation and in turn, the severity of disease. In acute viral 113 infection, TLR stimulation and TNF signaling induced myeloid cell aggregation 114 in the liver(51). These myeloid aggregates induced localized CD8 T cell 115 proliferation. Myeloid cell aggregation was not observed in chronic viral 116 infection. It is yet to be determined if immune cell aggregation occurred in non- 117 viral induced liver injury. The molecules associated with immune aggregation 118 are also unknown. Aggregation of leukocytes within the liver tissue during 119 disease has been most observed in HCV and ALD, though their role in 120 pathogenesis and mode of development is still unclear(52). We have previously 121 shown that CD147 is increased in cirrhotic liver and expressed by hepatocytes 122 (not HSC) during progressive liver injury(15). Herein we report CD147 123 upregulation on multiple immune cells during liver injury. This manuscript is the 124 first description of a novel CD147 mediated mechanism of immune cell 125 aggregation in liver injury. Specifically, we have shown that CD147 mediated 126 immune cell aggregation is independent of cell proliferation and anti-CD147 127 intervention reduces immune cell aggregation. 128 129 130 131 132 133 134 135 136 bioRxiv preprint doi: https://doi.org/10.1101/600676; this version posted April 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 5 137 Materials and Methods 138 139 Human ethics 140 141 Human tissues samples were obtained from Royal Prince Alfred Hospital, 142 Sydney with approval of Human Research Ethics Committee (X10-0072). 143 Human tissue used in this study was previously utilized for research(12, 15). 144 Informed written consent was obtained from all participants. The ethics 145 committee waived the need for written consent for use of donor tissue. In 146 Australia, the ethics of human research is governed by the National Statement 147 on Ethical Conduct in Human Research (2007) issued by the National Health 148 and Medical Research Council (NHMRC). Under these guidelines all research 149 involving humans requires ethical approval. Non-diseased donor and end- 150 stage cirrhotic liver tissues were collected from patients attending Prince 151 Alfred Hospital, Sydney during liver transplantation. 152 153 Mice 154 Mice wild type (balb/c and C57Bl/6) were housed at the Centenary Institute 155 Animal facility in compliance with the Animal Care and Ethics Committee at the 156 University of Sydney (K75/10-2008/3/4801).