Fig 2: Effects of neutrophil depletion on cancer progression. (A) Murine ID8-KRAS cells (2×106) were intraperitoneally injected into 31 mice (iso-type, n=15; and anti-Ly6G, n=16). The mice were intraperitoneally injected with 250 µg of anti-Ly6G mAb or matched isotype in 0.5 ml of PBS every 3 days from day 7 after inoculation of ID8-KRAS cells. For the mice survival analysis, the end point was determined as 'body weight (BW) = 23 g', and mice were sacrificed when their BW exceeded 23 g after inoculation. The log-rank test was used to detect differences (Kaplan-Meier survival curves). (B) ID8-KRAS-injected mice were treated with anti-Ly6G mAb (n=9) or isotype control mAb (n=8) from day 10 after the inoculation of ID8-KRAS cells, and the ascites were obtained on day 16 after washing with 3 ml of PBS. The ascites from no cancer mice were also obtained after washing with 3 ml of PBS (n=6). The interleukin (IL)-6 concentration in ascites of the 3 groups (no cancer, ID8-KRAS treated with isotype mAb, and ID8-KRAS treated with anti-Ly6G mAb) was analyzed using specific ELISA kits. Error bars represent the means ± SEM. Statistical analysis was performed using the Student's t-test. The P-value was adjusted using the Holm's method (*P<0.05).

Fig 3: Chil1 was positively correlated with Ly6g in the injured liver of mice and could be secreted by A-Neu. (A) The mRNA expression of Chil1 was examined by RT-qPCR in the injured liver of MCDHF-treated mice in indicated times (n=6). (B) S1P stimulated neutrophils for 2 hours, then PBS washed the cells, and continued to culture for 6 hours, the secretion of CHIL1 in cell supernatant was detected by ELISA, n=4. (C) Correlation analysis between Chil1 mRNA expression with neutrophil marker Ly6g, macrophage marker Adgre1, hepatocyte marker Alb and myofibroblast marker Acta2 in the injured liver of MCDHF-treated mice. Nonparametric test (Kruskal-Wallis test) was used in (A) Student’s t test was used in (B) Pearson’s test was used in (C) *P < 0.05 vs. MCDHF treated group for 0 days or control. MCDHF, methionine-choline-deficient and high-fat diet; S1P, Sphingosine 1-phosphate; Chil1, Chitinase-like 1; Ly6G, lymphocyte antigen 6 complex, locus G; Adgre1, adhesion G protein-coupled receptor E1; Acta2, actin alpha 2; Alb, albumin.

Fig 4: Effects of neutrophil depletion on the T cell subset. ID8-KRAS-injected mice were treated with anti-Ly6G mAb (n=9) or isotype control mAb (n=8) from day 10 after the inoculation of ID8-KRAS cells and sacrificed on day 16. No cancer mice were used as controls (n=6). Ascites and spleen were obtained from the no-cancer mice and ID8-KRAS mice treated with anti-Ly6G or isotype mAb after washing with 3 ml of PBS. T cells were collected by Percoll density gradient (44%/70%). T cells were stained with the following antibodies: PE-Cy5-anti-mouse CD3e, FITC-anti-mouse CD8a, and APC-anti-mouse CD4. The (A) CD4/CD3e ratio of ascites and spleen and (B) CD8/CD3 e ratio of ascites and spleen were analyzed by flow cytometry. Error bars represent the means ± SEM. Statistical analysis was performed using the Student's t-test. The P-value was adjusted using the Holm's method (*P<0.05, **P<0.01).

Fig 5: Infusion of A-Neu ameliorated MCDHF-induced liver injury in mice. (A) Schematic diagram of experimental design of tail vein injection of neutrophils (5×106 cells/mouse) after 2 weeks of MCDHF diet in mice. (B) Representative flow cytometry analysis of EGFP+ Ly6G+ neutrophils on CD45+ CD11b+ monocytes in the liver of mice in 4 hours or 12 hours after injection of EGFP+ N-Neu or EGFP+ A-Neu. (C) Infusing N-Neu or A-Neu into mice after 2 weeks of MCDHF diet and harvesting liver in 4 days after injection. The representative H&E staining images of liver sections were performed, inflammatory area was indicated by circle. (D) Areas of Inflammation were quantified, (control group, n=3; MCDHF group, n=6). (E) Serum ALT levels were measured in indicated groups, n=5 each group. The results were presented as mean ± SD. One-way ANOVA was used. *P < 0.05. Scale bar = 100µm. MCDHF, methionine-choline-deficient and high-fat diet; A-Neu, activated neutrophil; N-Neu, non-activated neutrophil; ALT, alanine aminotransferase; EGFP, enhanced green fluorescence protein.