Intravital imaging of splenic classical monocytes modifying the hepatic CX3CR1+ cells motility to exacerbate liver fibrosis via spleen-liver axis

CX3CR1+ cells play a crucial role in liver fibrosis progression. However, changes in the migratory behavior and spatial distribution of spleen-derived and hepatic CX3CR1+ cells in the fibrotic liver as well as their influence on the liver fibrosis remain unclear. Methods: The CX3CR1GFP/+ transgenic mice and CX3CR1-KikGR transgenic mice were used to establish the CCl4-induced liver fibrosis model. Splenectomy, adoptive transfusion of splenocytes, in vivo photoconversion of splenic CX3CR1+ cells and intravital imaging were performed to study the spatial distribution, migration and movement behavior, and regulatory function of CX3CR1+ cells in liver fibrosis. Results: Intravital imaging revealed that the CX3CR1GFP cells accumulated into the fibrotic liver and tended to accumulate towards the central vein (CV) in the hepatic lobules. Two subtypes of hepatic CX3CR1+ cells existed in the fibrotic liver. The first subtype was the interacting CX3CR1GFP cells, most of which were observed to distribute in the liver parenchyma and had a higher process velocity; the second subtype was mobile CX3CR1GFP cells, most of which were present in the hepatic vessels with a faster moving speed. Splenectomy ameliorated liver fibrosis and decreased the number of CX3CR1+ cells in the fibrotic liver. Moreover, splenectomy rearranged CX3CR1GFP cells to the boundary of the hepatic lobule, reduced the process velocity of interacting CX3CR1GFP cells and decreased the number and mobility of mobile CX3CR1GFP cells in the fibrotic liver. Transfusion of spleen-derived classical monocytes increased the process velocity and mobility of hepatic endogenous CX3CR1GFP cells and facilitated liver fibrosis progression via the production of proinflammatory and profibrotic cytokines. The photoconverted splenic CX3CR1+ KikRed+ cells were observed to leave the spleen, accumulate into the fibrotic liver and contact with hepatic CX3CR1+ KikGreen+ cells during hepatic fibrosis. Conclusion: The splenic CX3CR1+ monocytes with classical phenotype migrated from the spleen to the fibrotic liver, modifying the migratory behavior of hepatic endogenous CX3CR1GFP cells and exacerbating liver fibrosis via the secretion of cytokines. This study reveals that splenic CX3CR1+ classical monocytes are a key driver of liver fibrosis via the spleen-liver axis and may be potential candidate targets for the treatment of chronic liver fibrosis.


Figure S1 .
Figure S1.Flow cytometry analysis of splenic CX3CR1 + cells and their phenotypic characteristics in Oil/CCl4-treated mice.(A) Spleen in the Oil and CCl4-treated mice.(B) Spleen/body weight ratio was calculated (n = 6 mice per group).(C) The percentage of splenic CX3CR1 + cells in Oil-treated mice or CCl4-treated mice (n = 3 mice per group).(D) The number of splenic CX3CR1 + cells in Oil-treated mice or CCl4-treated mice (n = 3 mice per group).(E) The phenotypic characteristics of splenic CX3CR1 + cells in Oil-treated mice or CCl4-treated mice (n = 3 mice per group).Data are presented as the mean ± SEM.

Figure S2 .
Figure S2.Flow cytometry analysis of hepatic CX3CR1 + cells and their subsets in Oil/CCl4treated mice with or without splenectomy.(A) Gating strategy of hepatic CD45 + cells.(B) Gating strategy of hepatic CX3CR1 + cells and their subsets.

Figure S3 .
Figure S3.Phenotypic characteristics of CX3CR1 + cells and their subsets in the livers of CCl4treated mice with or without splenectomy.(A) Phenotypic analysis of hepatic CX3CR1 + cells from CCl4-treated mice by flow cytometry.(B-D) The hepatic CX3CR1 + cells and their subsets have distinct phenotypic profiles.(E-G) Phenotypic analysis of hepatic CD11b + CX3CR1 + cells and their subsets from fibrotic mice at one week after splenectomy by flow cytometry (n = 3 mice per group).Data are presented as the mean ± SEM.

Figure S4 .
Figure S4.The distribution of CX3CR1 GFP cells in each distribution index (r) from different groups.(A) The distribution of CX3CR1 GFP cells in each distribution index (r) from Oil-treated group with Spx; the bin value is 0.04.(B) The distribution of CX3CR1 GFP cells in each distribution index (r) from CCl4-treated group with Sham; the bin value is 0.04.(C) The distribution of CX3CR1 GFP cells in each distribution index (r) from CCl4-treated group with Spx; the bin value is 0.04.

Figure S5 .
Figure S5.Intravital imaging of hepatic CX3CR1 GFP cell subtypes in the Oil/CCl4-treated mice with or without splenectomy.(A) Representative fluorescence images of CX3CR1 GFP cells

Figure S6 .
Figure S6.The different mobile type of hepatic CX3CR1 GFP cell in the Oil/CCl4-treated mice with or without splenectomy.(A) Representative images showing the migratory trajectory of mobile CX3CR1 GFP cell with mean velocity less than 2 μm/min ("stable" type) over a 20-minute period.Green: CX3CR1 GFP cells; Red: AF647 anti-CD31 labeled hepatic vessels.White dotted line shows the vessels.Scale bar, 5 μm.(B) Representative images showing the migratory trajectory of mobile CX3CR1 GFP cell with mean velocity between 2-3 μm/min ("confined" type) over a 20minute period.Green: CX3CR1 GFP cells; Red: AF647 anti-CD31 labeled hepatic vessels.White dotted line shows the vessels.Scale bar, 5 μm.(C) Representative images showing the migratory trajectory of mobile CX3CR1 GFP cell with mean velocity more than 3 μm/min ("serial" type) over a 20-minute period.Green: CX3CR1 GFP cells; Red: AF647 anti-CD31 labeled hepatic vessels.White dotted line shows the vessels.Scale bar, 15 μm.(D) The relative fraction of the different mobile type of CX3CR1 GFP cells in the liver of Oil/CCl4-treated mice with or without splenectomy (n = 22-28 fields, from 3 mice per group).(E) The number of the different mobile type of CX3CR1 GFP (per field) in the liver of Oil/CCl4-treated mice with or without splenectomy (n = 22-28 fields, from 3 mice per group).

Figure S7 .
Figure S7.Phenotypic characteristics of mobile CX3CR1 GFP cells and interacting CX3CR1 GFP cells in the livers of CCl4-treated mice with or without splenectomy.(A-B) The surface marker profile of hepatic mobile CX3CR1 GFP cells and interacting CX3CR1 GFP cells.(C) Phenotypic analysis of hepatic mobile CX3CR1 GFP cells and interacting CX3CR1 GFP cells from fibrotic mice at

Figure S8 .
Figure S8.Intravital imaging of CX3CR1 GFP cells in the livers of CCl4-treated mice with or without splenectomy at 14 days after splenectomy.(A) Schedules of generation of liver fibrosis model, the splenectomy and intravital imaging.(B) Representative fluorescence images of the CX3CR1 GFP cells in the livers of CCl4-treated mice with or without splenectomy.Green: CX3CR1 GFP cells; Red: AF647 anti-CD31 labeled hepatic vessels.Scale bar, 50 μm.(C) The positive areas of GFP were quantified using Image J software (from 3 mice per group).(D) The velocity of interacting CX3CR1 GFP cells processes in the liver parenchyma.Each dot represents a single cell process, and the red bars indicate mean values (3 mice per group).(E) The cell number of mobile CX3CR1 GFP cells in the hepatic vessels of CCl4-treated mice with or without splenectomy (n = 21-22 fields, from 3 mice per group).(F-H) Scatter plots of mean velocity (F), arrest coefficient (G), and confinement ratio (H) of mobile CX3CR1 GFP cells in the hepatic vessels of CCl4-treated mice with or without splenectomy (n = 21-22 fields, from 3 mice per group).Each dot represents a

Figure S9 .
Figure S9.Flow cytometry analysis and immunofluorescence staining to identify splenic CX3CR1 + cells and their subsets in the spleen of Oil/CCl4-treated mice.(A) Gating strategy of splenic classical monocytes and non-classical monocytes.(B) The percentage of CD11b + CX3CR1 + cells, classical monocytes and non-classical monocytes out of splenocytes in the fibrotic spleen (n = 3 mice per group).(C-D) The percentage of sorted splenic CD11b + CX3CR1 + cells (C), CD11b + CD115 + CX3CR1 low Ly6C high cells and CD11b + CD115 + CX3CR1 high Ly6C low cells (D).(E) The cell surface receptor profile of CX3CR1 GFP cells in the marginal zone and subcapsular red pulp of the fibrotic spleen was examined by immunofluorescence staining.Green: CX3CR1 GFP cells; Blue: DAPI; Magenta: CD68/ F4/80.Scale bar (large image), 50 μm.Scale bar (small image), 10 µm.White arrows indicate CX3CR1 GFP cells.

Figure S10 .
Figure S10.Phenotypic characteristics of splenic CX3CR1 + cells and their subsets from Oil/CCl4-treated mice by flow cytometry.(A-C) The splenic CX3CR1 + cells and their subsets have distinct phenotypic profiles.(D-E) Phenotypic analysis of CD11b + CX3CR1 + cells and their subsets in the spleen of Oil and CCl4-treated mice by flow cytometry (n = 3 mice per group).Data are presented as the mean ± SEM.

Figure S11 .
Figure S11.Intravital imaging of the spatial distribution of CX3CR1 GFP cells in the fibrotic liver after adoptively transferring splenic cells.(A) Intravital imaging of CX3CR1 GFP cells distribution in the hepatic lobule.Green: CX3CR1 GFP cells; Red: AF647 anti-CD31 labeled hepatic vessels; Blue: CX3CR1 GFP cells in the hepatic lobule; Magenta: Hepatic lobule.Scale bar, 50 µm.(B) The density of CX3CR1 GFP cells in one hepatic lobule (n = 10-21, from 3 mice per group).Data are presented as mean ± SEM. (C-F) The distribution of CX3CR1 GFP cells in each distribution index from different groups; the bin value is 0.04.(G) The distribution curves of CX3CR1 GFP cells in each distribution index from different groups; the bin value is 0.005.(H) The distance of CX3CR1 GFP cells to the hepatic central vein (CV) in the hepatic lobules.

Figure S13 .
Figure S13.Intravital imaging revealed the localization and migration behavior of adoptive splenic cells in the fibrotic liver after adoptively transferring splenic cells.(A) The distribution of adoptive splenic classical monocytes in the hepatic lobules.Green: CX3CR1 GFP cells; Blue: Classical monocytes; Red: AF647 anti-CD31 labeled hepatic vessels; Magenta: Boundary of the hepatic lobules.Scale bar, 100 μm.High-magnification views of the adoptive splenic classical monocytes in the liver are shown on the right.Scale bar, 10 µm.(B) The trajectory of adoptive splenic cells in different groups.

Figure S14 .
Figure S14.The phenotypic characteristics of neighboring endogenous CX3CR1 GFP cells contacted with adoptive splenic cells in the fibrotic liver.(A) Phenotypic characteristics of neighboring endogenous CX3CR1 GFP cells contacted with adoptive splenocytes from healthy mice.Scale bar (large image), 25 μm.Scale bar (small image), 8 μm.(B) Phenotypic characteristics of neighboring endogenous CX3CR1 GFP cells contacted with adoptive splenic CX3CR1 + cells from

Figure S15 .
Figure S15.Intravital imaging revealed the migration behavior of hepatic endogenous CX3CR1 GFP cells in the fibrotic liver after adoptively transferring splenic cells.(A) The trajectory of interacting CX3CR1 GFP cells in the liver parenchyma.(B) The trajectory of mobile CX3CR1 GFP cells in the hepatic vessels.

Figure S16 .
Figure S16.Intravital imaging revealed the migration characteristics of hepatic endogenous CX3CR1 cells and adoptive splenic cells.(A-C) Scatter plots of mean velocity of endo-mobile CX3CR1 GFP cells and adoptive splenic cells in different groups (3 mice per group).(D-F) Scatter plots of arrest coefficient of endo-mobile CX3CR1 GFP cells and adoptive splenic cells in different groups (3 mice per group).(G-I) Scatter plots of confinement ratio of endo-mobile CX3CR1 GFP cells and adoptive splenic cells in different groups (3 mice per group).

Figure S17 .
Figure S17.The effect of the procedure of CX3CR1-KikGR on cell viability and the stability of KikRed protein levels in CX3CR1 + cells.(A) Immunofluorescence imaging of unconverted and photoconverted spleen.Scale bar, 100 µm.(B) The viability of splenocytes from unconverted and photoconverted CX3CR1-KikGR transgenic mice was assessed using the fixable viability dye eFluor506 and analyzed by flow cytometry immediately (0 h) or 24 h after photoconversion (n = 3 mice per group).(C) Mouse peritoneal macrophages isolated from unconverted CX3CR1-KikGR transgenic mice were photoconverted by exposure to the 405 nm light (cells was exposed for 3 minutes, 200 mW/cm 2 ) in vitro and analyzed by confocal imaging immediately (0 h) or after culture for 24h, 48h, 72h and 144 h at 37 °C.Scale bar, 50 µm.(D) The