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Fig. 3 | Journal of Biomedical Science

Fig. 3

From: Cholestasis-induced phenotypic transformation of neutrophils contributes to immune escape of colorectal cancer liver metastasis

Fig. 3

Primary bile acids contribute to the immunosuppressive phenotype of neutrophils. A, B C57BL/6 mice received BDL surgery and liver capsule injection of MC38 cells simultaneously on day 0, and a 400 μg anti-Ly6G i.p. injection on day 1, 2, 4, and 6, and were euthanized on day 8. Representative images of LM (A) and diameters of LM (B) of MC38-liver capsuled injected BDL mice after receiving anti-Ly6G treatment (n = 9 and 7 for the control and anti-Ly6G group, respectively). C Kaplan–Meier analysis of MC38-liver capsuled injected BDL mice with or without anti-Ly6G treatment (Log-rank test, n = 5 per group). D Bile acids from the liver (left) and serum (right) were analyzed by LC-MS. Hierarchical cluster analysis was performed on differential bile acid metabolites. TCA, Taurocholic acid; GCA, Glycocholic acid; Tβ-MCA, Tauro-β-muricholic acid; GLCA-3S, Glycolithocholic acid-3-sulfate; β-MCA, β-muricholic acid; TCDCA, Taurochenodeoxycholic acid; TUDCA, Tauroursodeoxycholic acid; NCA, Norcholic acid; UDCA, Ursodeoxycholic acid; MDCA, Murideoxycholic acid; 3-oxo-CA, 3-Oxocholic acid; TLCA, Taurolithocholic acid; DCA, Deoxycholic acid. E Flow cytometric analysis and quantitative results of Arg1 expression of neutrophils under 24h of TCA or Tβ-MCA stimulation (n = 6 per group). F Immunofluorescence images and quantification of expression of iNOS and Arg1 of neutrophils after Tβ-MCA stimulation. G The mRNA level of immunosuppressive genes of neutrophils after Tβ-MCA stimulation. H Schematic diagram of the co-culture system for neutrophils and lymphocytes. Isolated neutrophils were treated with bile acids and cocultured with CD3/CD28-primed CD8+ T cells for 24 h. Then, lymphocytes were isolated for the tumor cell killing assay and flow cytometry analysis. I Flow cytometric analysis and quantitation of IFN-γ, GZMB, and PD-1 expression of CD8+ T cells co-cultured with neutrophils pretreated with Tβ-MCA (n = 4 and 3 for the control and Tβ-MCA groups, respectively). GZMB, Grazyme-B. J Flow cytometric analysis of the proliferation of mouse CD8+ T cells co-cultured with Tβ-MCA-pretreated neutrophils. K The mRNA level of leukocyte differentiation-related genes of mouse CD8+ T cells after co-culturing with Tβ-MCA-pretreated neutrophils. Data represent mean ± SEM of 3 biologically independent experiments (Two-tailed t test). ns, no significance. L Flow cytometric analysis of apoptosis of MC38 cells induced by CD8+ T cells co-cultured with Tβ-MCA-pretreated neutrophils (n = 3 per group). Data represent mean ± SEM of 3 biologically independent experiments (Two-tailed t test). ns, no significance; Sham, sham surgery; BDL, bile duct ligation surgery

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Journal of Biomedical Science

ISSN: 1423-0127