CD147 mediates intrahepatic leukocyte aggregation and determines the extent of liver injury

Abstract

Background: Chronic inflammation is the driver of liver injury and results in progressive fibrosis and eventual cirrhosis with consequences including 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 a mAb targeting CD147. Inflammatory cell subsets were assessed by multiparameter flow cytometry.

Results: In liver injury, we observe abundant, intrahepatic leukocyte clusters defined as ≥5 adjacent CD45+ cells which we have termed "leukocyte aggregates". We have shown that these leukocyte aggregates have a significant effect 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 is accompanied by no change in overall intrahepatic leukocyte numbers. Further, blocking of 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. Here we demonstrate that leukocyte aggregates determine the extent of liver injury.

Fig 1. Quantitative real-time PCR for CD147 expression in a mouse model of TAA-induced liver injury.

(A) CD147 expression in whole liver from control, 4 week and 20-week TAA-treated mice. (B) Comparison of CD147 expression in liver leukocytes and hepatocytes throughout the course of TAA-induced injury. (C) CD147 expression in blood, liver, and spleen at 20 weeks compared to control. Data were normalised to 18S and expressed as fold change from control. Bars represent mean +SEM. Mann-Whitney test was performed to assess significance from control where * p<0.05 (n = 5–7 per group).

Fig 2. CD147 surface expression on leukocyte subsets isolated from livers in progressive liver injury.

Flow cytometry was performed on liver leukocytes from control and TAA-treated mice. Each leukocyte subset was analysed for expression of CD147 and data recorded as the percentage of cells positive for CD147 (%CD147+) or mean fluorescence intensity (MFI). Representative histogram plots are shown for each subset, where the black histogram represents the indicated liver subset and grey histogram indicates isotype control. Bar graphs represent mean ± SEM Mann-Whitney test was performed to assess significance from control where * p<0.05, **p<0.01, ***p<0.001 (n = 5–8 per group).

Fig 3. Aggregation of immune cell infiltrate in mouse liver injury.

Inflammatory infiltrates in liver tissue of control, 4, 8 and 20-week TAA-treated C57Bl/6 mice and control, 1 day and 4 weeks CCl₄ treated C57Bl/6 and BALB/c mice. Representative images of CD45 stained liver sections from (A) non-injured control; (B) 20-week TAA and (C) 4 week CCl₄ treated mice. Scale represents 100μM. (D) Total CD45⁺ cells were quantified per field of view with 4–10 fields counted per section. Bars represent mean+SEM. (E) Immune cell aggregates were quantified (≥5 CD45⁺ cells per cluster) per field of view (FOV). Bars represent mean+SEM. Mann-Whitney test was performed to assess significance from control where * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (n = 5–8 per group).

Fig 4. Aggregation of CD147+ immune cells in diseased liver.

Representative images of liver sections stained with CD45 (red) and CD147 (green) from (A) healthy control and (B) diseased patient liver (autoimmune hepatitis) on either a 20x (left panels) or 63x (right panels) objective. Yellow indicates co-expression of CD147 and CD45. Scale bar represents 100μM. Quantification of (C) total CD45+ cells and (D) immune aggregates in healthy, PSC, EtOH, AIH and HCV livers. Bars represent mean+SEM. Unpaired t-test was performed to assess significance from control where * p<0.05 and **p<0.01 (n = 4–15 per group). Donor = healthy control liver, PSC = primary sclerosing cholangitis, ETOH = alcohol-induced liver damage, AIH = Autoimmune hepatitis, HCV = hepatitis C virus liver injury.

Fig 5. CD147 blockade prevents immune cell aggregation in liver injury.

Anti-CD147 antibody intervention in CCl₄ induced liver injury in C57Bl/6 (left panels) or BALB/c (right panels) mice. CD45+ leukocytes were stained in frozen tissue sections in control, treatment (CCl₄), treatment with anti-CD147 antibody intervention, or treatment with IgG2a isotype antibody controls. Representative immunofluorescent images of CD45⁺ clusters defined as ≥5 cells per cluster (white circles) are shown in C57Bl/6 (A) and BALB/c mice (B) treated with IgG2a. Representative immunofluorescence images of CD45⁺ clusters following αCD147 intervention in C57Bl/6 (C) and BALB/c (D) mice. Total CD45⁺ leukocytes in C57Bl/6 (E) and BALB/c (F) mice. (G) Percentage of CD45⁺ cells in aggregates for each treatment group in C57Bl/6 (G) and BALB/c (H) mice. ALT levels (U/L) as a measure of liver damage in C57Bl/6 (I) and BALB/c (J) mice, calculated as a fold change from control. Bar graphs represent mean ± SEM. Mann-Whitney test was performed to assess significance from control where * p<0.05, **p<0.01, ***p<0.001 (n = 5–8 per group). Control = untreated, CCl₄ injury = injury alone, αCD147 = anti-CD147 mAb in CCl₄ injury, IgG2a = Isotype mAb control in CCl₄ injury.

Fig 6. Anti-CD147 intervention reduces aggregation of macrophages, B cells, T cells and granulocytes during liver injury.

CCl₄ treated C57Bl/6 mice liver tissue was stained for CD45 colocalisation with (A) F4/80, (B) B220, (C) Gr1 or (D) CD3, to identify immune subset specific aggregates. Representative images (left panel) and quantified immune cell-specific aggregates (right panel; calculated as at least one cell of interest per aggregate) in mice treated with CCl₄ alone or CCl₄ in conjunction with anti-CD147 intervention. Bar graphs represent mean ± SEM. Mann-Whitney test was performed to assess significance from control where * p<0.05, **p<0.01 (n = 5–7 per group). CCl₄ = injury alone, αCD147 = anti-CD147 in CCl₄ injury.