The Role of CD147 in Leukocyte Aggregation in Liver Injury

Background Chronic inflammation is the driver of liver injury resulting in progressive fibrosis and eventual cirrhosis. The consequences include both liver failure and liver cancer. We have previously described increased expression of the highly multifunctional glycoprotein CD147 in liver injury. This work describes a novel role of CD147 in liver inflammation and the importance of leukocyte aggregates in determining the extent of liver injury. Methods Non-diseased, progressive injury and cirrhotic liver from humans and mice were examined using mAb targeting CD147. Inflammatory cell subsets were assessed by multicolor flow cytometry. Results In liver injury, we observe abundant intrahepatic leukocyte clusters defined as ≥5 adjacent CD45+ cells which we have labelled “leukocyte aggregates”. We have shown that these leukocyte aggregates are significant in determining the extent of liver injury. If CD147 is blocked in vivo, these leukocyte aggregates diminish in size and number together with a marked significant reduction in liver injury including fibrosis. This accompanied by no change in overall intrahepatic leukocyte numbers. Further, blocking aggregation formation occurs prior to an appreciable increase in inflammatory markers or fibrosis. Additionally, there were no observed, “off-target” or unpredicted effects in targeting CD147. Conclusion CD147 mediates leukocyte aggregation which is associated with the development of liver injury. This is not a secondary effect, but a cause of injury as aggregate formation proceeds other markers of injury. Leukocyte aggregation has been previously described in inflammation dating back over many decades but till now been shown to determine the extent of injury.


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)(3)(4)(5)(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)(8)(9)(10)(11) .

81
Importantly, we have previously identified a number of novel pathways 82 of liver injury (12)(13)(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) .

143
Human tissue used in this study was previously utilized for research (12,15) .

144
Informed written consent was obtained from all participants.

223
Statistical analysis was performed using Prism 6. Mann-Whitney U t-test was 224 performed to compare against control, unless otherwise specified.

225
Significance was accepted at p<0.05. Data is shown as mean ± SEM. As

226
indicated some data is expressed as fold change from control. in the expression of CD147 mRNA in whole liver tissue ( Fig 1A). When 249 hepatocytes and leukocytes were analysed separately a significant increase of 250 CD147 mRNA expression was observed in leukocytes (30-fold) ( Fig 1B).

252
shows that CD147 expression is upregulated in a mouse model of liver injury 253 and CD147 predominantly increased on leukocytes, not hepatocytes following

270
We next characterized the expression of CD147 on various leukocyte subsets 271 during the course of liver injury (Fig 2) increased on all liver leukocytes, with peak expression at 8 weeks of injury.

277
Interestingly, as liver injury progressed, we observed a significant loss of NKT 278 cells and macrophages and a significant increase of CD8 + T cells, eosinophils 279 and neutrophils from the liver.

281
To establish whether similar CD147 expression patterns were occurring in an 282 extrahepatic lymphoid organ, the spleen was also analysed by flow cytometry 283 in the same manner described above. This trend was true for all sub-

307
Aggregation of lymphocytes during liver injury in mouse models.

308
To determine how lymphocytes localized during chronic liver injury, CD45 309 staining was conducted on a range of injury models including TAA treatment in 310 C57Bl/6, CCl 4 in C57Bl/6 and CCl 4 in balb/c mice (Fig 3). In uninjured control 311 livers, lymphocytes dispersed evenly within the liver tissue (Fig 3A). In contrast,

312
in both TAA ( Fig 3B) and CCl 4 (Fig 3C) injury models not only were total CD45 313 cells increased (Fig 3D) but CD45 cells were found to aggregate (defined as >5

314
CD45 + cells in contact). Importantly the majority of aggregation occurred  Aggregation of lymphocytes in injured human liver.

342
To confirm that immune cell expression of CD147 and immune cell aggregation 343 was not exclusive to mouse models we examined human liver tissue from 344 healthy donors ( Fig 4A) and patients with PSC, alcohol induced liver damage 345 (EtOH), AIH and HCV (Fig 4B). Interestingly we not only observed CD147

372
To determine if CD147 was important for the formation of immune cell 373 aggregates the effects of anti-CD147 intervention were studied in CCl 4 374 induced liver injury in both C57Bl/6 and balb/c mice. Anti-CD147 did not significantly reduce the number of CD45 + cells in the liver (Fig 5E, F). The

percentage of immune cells that formed immune cell aggregation was
377 reduced with anti-CD147 intervention in both mouse backgrounds (Fig 5G, H).

378
In C57Bl6 there was non-significant, less than 1.5-fold, increase in ALT with 379 injury and no significant change was seen with anti-CD147 intervention (Fig   380  5I). However, in balb/c mice with more significant injury the reduction in 381 immune cell aggregation correlated with as significantly reduced serum ALT 382 levels ( Fig 5J). Thus, CD147 inhibition appeared to significantly reduce the 383 formation of immune cell aggregates and reduce significant liver injury. aggregates during liver injury (Fig 6). After anti-CD147 treatment, we observed 412 a decrease in the number of aggregates with at least one F4/80+, B220+ and 413 CD3+ cell. The number of Gr-1+ cells in aggregates was not altered after anti-

419
CCl 4 treated C57Bl/6 mice liver tissue was stained for CD45 colocalisation with This study has demonstrated that with progressive inflammation-430 associated tissue injury, immune cells cluster and contribute directly to the 431 magnitude of the injury. Our novel discovery is that with liver injury CD147 is 432 increased on the surface of leukocytes and then mediates cell-cell aggregation 433 that determines the extent of liver injury. However, if we blocked CD147 with a 434 mAb then CD45 + cell numbers in the liver remain unchanged but leukocytes 435 are no longer found in aggregates. Importantly, we have already reported there 436 is a significant reduction in liver injury seen with anti-CD147 mAb (15) . Further, 437 CD147 mediated leukocyte aggregation appears to cause or significantly 438 exacerbate injury as aggregate formation proceeds the development of 439 significant fibrosis (15) . Therefore, this is not just a reduction in aggregation and 440 inflammatory markers (AST/ALT) but also a reduction in resultant fibrosis.

441
All intrahepatic leukocyte subpopulations (CD4 + , CD8 + , NK, B-cell and 442 macrophages) rapidly increase CD147 surface protein and total mRNA 14 443 expression with liver injury. Therefore, this data shows that following liver injury, 444 circulating and intrahepatic leukocytes increase CD147 expression.