Figure 5: Tim-3 inhibits the HO-1-IL-1β signaling pathway in macrophages via Nrf2.

(A) Peritoneal macrophages isolated from WT and Tim-3-TG mice were analyzed for HO-1 mRNA levels by real-time-PCR. (B) Control or RAW264.7 cells silenced for Nrf2 as in Fig. 2A were examined for HO-1 mRNA levels by real-time PCR. (C) RAW264.7 cells or Nrf2 knockdown RAW264.7 cells were incubated with 20 μg/ml of sTim-3-Trx (sTim-3) or Trx control (Con) for 12 h, then were collected and HO-1 mRNA levels examined by real-time PCR. (D) RAW264.7 cells were incubated with 20 μg/ml of sTim-3-Trx (sTim-3) or Trx control (Con) in the presence of PR619 (20 μM) or vehicle (DMSO) for 12 h, then were collected and HO-1 mRNA levels examined by real-time PCR. (E) RAW264.7 cells were incubated with 20 μg/ml of sTim-3-Trx (sTim-3) or Trx control (Con) in the presence or absence of an HO-1 inhibitor (Snpp-IX, 5 μM) or vehicle (DMSO) for 12 h, then were infected with L. monocytogenes (1 × 10⁶ CFU) for 12 h and IL-1β mRNA levels examined by real-time PCR. (F,G) Tim-3 blockade promotes macrophages killing of L. monocytogenes. Raw264.7 cells were infected with L. monocytogenes (F) or CFSE-labeled L. monocytogenes (G) for 2 h, then washed, and incubated with Ampicillin to kill extracellularly located bacteria in the presence of sTim-3 or control protein for 24 h. Macrophages were either homogenized for CFU counting (F) or were imaged using confocal laser scanning microscope to examine the living bacteria (CFSE-bright spot) (G). The data are expressed as the mean ± SD for triplicate wells; **p < 0.01; ***p < 0.001, and are representative of the results obtained in three independent experiments.