Figure 4: Tim-3 inhibits macrophage control of L. monocytogenes via the Nrf2-CD36 signaling axis.

(A) Peritoneal macrophages isolated from WT or Tim-3-TG mice were examined for CD36 expression by flow cytometry. (B,C) RAW264.7 cells (NC) or Nrf2-knockdown (Nrf2-si) RAW264.7 cells were incubated with 20 μg/ml of soluble sTim-3-Trx (sTim-3) or Trx control (Con) for 12 h (for PCR) or for 24 h (for FACS), then were collected and analyzed for CD36 mRNA expression by real-time PCR (B) and for CD36 protein expression (C) by flow cytometry. (D,E) RAW 264.7 cells were incubated with 20 μg/ml of sTim-3-Trx (sTim-3) or Trx control (Con) and either DMSO (vehicle) or the deubiquitination inhibitor PR619 (20 μM) for 12 h (for PCR) or for 24 h (for FACS), then were collected and analyzed for CD36 mRNA expression by real-time PCR (D) and for CD36 protein expression (E) by flow cytometry. (F,G) RAW264.7 cells infected with CFSE-labeled L. monocytogenes were incubated with PBS or the CD36 inhibitor SSO (20 μM) for 12 h in the presence of 20 μg/ml of sTim-3-Trx (sTim-3) or Trx control (Con), then were collected and analyzed for CFSE staining in macrophages by flow cytometry. (G) Representative data showing CFSE staining of the macrophages in (F). In (B,D,F), the results are expressed as the mean ± SD of triplicate wells. *p < 0.05; **p < 0.01; ***p < 0.001. The results shown are representative of those obtained in three independent experiments.