Fig 1.
CVC attenuates CCL2 and CCL5 dependent leukocyte chemotaxis in vitro.
(A) Representative FACS plots displaying transwell migration chemotaxis assays (at 2 hours) of CCL2-induced monocyte migration from total bone marrow of c57bl/6 wildtype mice. CVC was used at a concentration of 1μM. Normalized chemotaxis ratio towards CCL2 (5nM) or CCL5 (5nM) of bone-marrow derived monocytes compared to vehicle (Vhc). (B) Same analysis for NK cells from bone marrow. (C) Transwell migration assays using lymphocytes from mouse spleen. Normalized chemotaxis ratio of lymphocytes towards CCL2 (5nM) or CCL5 (5 nM). Data are presented as mean ± SD based on n≥3 per group. *p<0.05, **p<0.01, ***p<0.001 (unpaired Student t test).
Fig 2.
CVC reduces the accumulation of hepatic macrophages in CCl4-induced acute liver injury.
(A) Acute liver injury was conducted by a single IP injection of CCl4. The in vivo effects of CVC on immune cell migration into acutely injured liver was assessed at 12h, 24h and 36h after CCl4, and after one to three doses of CVC. (B) Liver histology (H&E staining) of representative liver sections for control and treatment groups. Hepatic injury is assessed by necrotic area fraction and serum alanine transaminase (ALT) levels. (C) Representative F4/80 immunohistochemical staining of liver sections and the corresponding F4/80 positive area fraction demonstrate reduced macrophage numbers in CVC treated livers. Data are presented as mean ± SD based on n≥3 per group. *p<0.05, **p<0.01, ***p<0.001 (unpaired Student t test).
Fig 3.
CVC inhibits hepatic monocyte infiltration in acute liver injury.
All results were obtained from c57bl/6 wildtype mice 36h after CCl4 challenge. (A) Representative FACS plots showing monocyte-derived macrophages (MoMF, dashed) and Kupffer cells (KC, solid) as well as corresponding quantification in percent of liver leukocytes. (B) Total numbers of blood monocytes and the Ly-6C positive subset were analyzed in parallel. (C+D) Unbiased t-SNE analysis of myeloid liver (C) or blood (D) cells from treatment groups (n = 6) illustrate myeloid immune cell populations, which are (mostly) unique in vehicle treated (yellow) or CVC treated mice (blue). Mixed cell population that are equally found in both treatment groups are displayed in dark-green (liver) or grey-blue (blood). Single cell clusters were further characterized by relative myeloid surface marker expression of treatment groups and compared to total liver or blood cells (representative histograms). Data are presented as mean ± SD based on n≥6 mice per group. *p<0.05, **p<0.01, ***p<0.001 (unpaired Student t test).
Fig 4.
CVC does not affect NK or T cell populations during acute liver injury in vivo.
All results were obtained from c57bl/6 wildtype mice 36h after CCl4 challenge. (A) Representative FACS plots and statistical summary showing liver NK cells, CD4 and CD8 T-cells. (B) Representative FACS plots and statistical summary of NK cells, CD4 and CD8 T-cells from blood. (C+D) Unbiased t-SNE analysis of lymphoid liver (C) or blood (D) cells from treatment groups (n = 6) illustrate immune cell populations, which are (mostly) unique in Vhc treated (yellow) or CVC treated mice (blue). Mixed cell population that are equally found in both treatment groups are displayed in dark-green (liver) or grey-blue (blood). Single cell clusters were further characterized by relative lymphoid surface marker expression of treatment groups and compared to total liver or blood cells (representative histograms). Data are presented as mean ± SD based on n≥6 mice per group. *p<0.05, **p<0.01, ***p<0.001 (unpaired Student t test).
Fig 5.
CVC modulates monocyte-dependent liver inflammation without directly interfering with macrophage polarization.
(A) RNA from whole liver tissue was subjected to quantitative gene expression analysis (NanoString kit, covering 72 genes). Gene expression analysis of macrophage (CCR2) and Kupffer cell (CD68) markers, hepatocyte function (albumin) as well as chemokines (CCL2) and inflammation associated genes (S100a8, S100a9). (B) Demonstration of Log2-fold change in gene expression of 19 chosen candidates comparing Vhc and CVC treated livers. (C) Bone marrow derived macrophages (BMDM) were cultured for 7 days and then stimulated for 24h with IFNγ (M1 phenotype), IL-4 (M2) or LPS (pathogen recognition), either in presence or without CVC (1μM). Representative phase contrast images of BMDM 24h after stimulation, taken at 10x, scale bar 100μm. Gene expression analysis of macrophage polarization markers and effector cytokines. Data are presented as mean ± SD based on n≥6 mice per group. *p<0.05, **p<0.01, ***p<0.001 (unpaired Student t test).