Objective: To investigate the meaning and molecular mechanisms of Galectin-9/T-cell immunoglobulin mucin-3 (Tim-3) pathway on lipopolysaccharide (LPS) induced murine macrophage M1/M2 subtype polarization.
Methods: The murine peritoneal macrophages RAW264.7 were cultured in vitro until the cells had matured with 80%-90% fusion rate. (1) The cells were cultured in serum-free medium and treated with 0 (blank control), 0.01, 0.1, 1, 10 and 100 mg/L LPS for 24 hours. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) or Western Blot was used to determine the expressions of M1 macrophage markers such as interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and M2 macrophage markers such as arginase-1 (Arg-1), leukocyte differentiation antigen 206 (CD206), as well as Tim-3 and Galectin-9 in the cells. (2) The other mice peritoneal macrophages were divided into blank control group (cultured in serum-free DMEM medium for 24 hours), LPS treatment group (cultured in serum-free DMEM medium containing 0.1 mg/L LPS for 24 hours) and α-lactose pretreatment group (pretreated with serum-free DMEM containing 40 μmol/L Galectin-9 signal antagonist 1 hour before LPS stimulation). Over closed Galectin-9 signal was used to verify the role of Galectin-9 in macrophage M1/M2 subtype polarization.
Results: (1) After stimulation with low concentrations of LPS (0.01 mg/L, 0.1 mg/L) for 24 hours, the expression of M1 markers was only slightly increased such as iNOS mRNA or not significantly changed such as IL-6 mRNA in macrophages, while the expressions of M2 markers such as Arg-1 mRNA and CD206 mRNA were significantly increased and peaked at LPS concentrations of 0.1 mg/L and 0.01 mg/L [compared with blank control group: Arg-1 mRNA (2-ΔΔCt) was 1.85±0.07 vs. 1.00±0.02, CD206 mRNA (2-ΔΔCt) was 2.03±0.11 vs.1.00±0.05, both P < 0.01]. With the increase of LPS concentration, the expressions of IL-6 mRNA and iNOS mRNA continued to increase, while the expressions of Arg-1 mRNA and CD206 mRNA were gradually decreased, and the macrophage M1/M2 subtype polarization status changed. At the same time, the level of Tim-3 protein in macrophages was significantly up-regulated after stimulation with 0.01 mg/L LPS as compared with that of blank control group (Tim-3/GAPDH: 0.84±0.04 vs. 0.69±0.02, P < 0.01), peaked at LPS concentrations of 0.1 mg/L, and then decreased with increasing LPS concentration. The intracellular Galectin-9 and supernatant secreted Galectin-9 (s-Galectin-9) protein levels showed no significant change after stimulation with low concentrations of LPS (0.01 mg/L, 0.1 mg/L), while then gradually decreased with the increase of LPS concentration. (2) Compared with blank control group, the mRNA expressions of M1 marker iNOS and M2 markers Arg-1 and CD206 were significantly increased in LPS treatment group, but IL-6 mRNA level was not changed significantly. The mRNA levels of IL-6 and iNOS were further elevated after pretreatment with α-lactose as compared with that of the LPS treatment group [IL-6 mRNA (2-ΔΔCt): 1.44±0.02 vs. 1.14±0.11, iNOS mRNA (2-ΔΔCt): 2.45±0.04 vs. 2.01±0.08, both P < 0.01], while the mRNA levels of Arg-1 and CD206 were significantly decreased [Arg-1 mRNA (2-ΔΔCt): 0.75±0.01 vs. 1.85±0.02, CD206 mRNA (2-ΔΔCt): 0.58±0.02 vs. 2.03±0.14, both P < 0.01]. Meanwhile, the blocking of Galectin-9 signaling could also reduce the extracellular s-Galectin-9 (compared with LPS treatment group: s-Galectin-9/GAPDH was 0.10±0.01 vs. 0.23±0.02, P < 0.01), down-regulated the expressions of Tim-3 and Galectin-9 (Tim-3/GAPDH: 0.28±0.01 vs. 0.43±0.01, Galectin-9/GAPDH: 0.21±0.01 vs. 0.43±0.01, both P < 0.01).
Conclusions: LPS regulates macrophage M1/M2 subtype polarization via Galectin-9/Tim-3 signaling pathway. Low-doses of LPS can limit the development of inflammation by accommodating the expression and secretion of Galectin-9 to polarize macrophages to M2. High-doses of LPS promotes the development of inflammation by down-regulating the expression and secretion of Galectin-9 to polarize macrophages to M1.