Fig 1: The mechanism that IL-37 suppresses M1 macrophage polarization. IL-37 suppresses M1 macrophage polarization through the Notch1-NF-?B axis pathways. The activation of Notch1 by TLR4 stimulation with LPS causes the generation of NICD1, which interacts with I?B and results in the activation of NF-?B, but the generation can be inhibited by IL-37. IL-37 prevents Notch1 and NF-?B activation and thus attenuates M1 polarization and inhibits the inflammatory response.
Fig 2: More M1 macrophages infiltrate in calcified aortic valves, with a reduction of IL-37 expression. (A) Normal and calcified human aortic valves were stained with HE. The arrow above shows the newly blood vessels in calcified aortic valves. Magnification, 200× and 400×; n = 6. (B–D) CD11c, CD206 and IL-37 expression are shown in calcified and non-calcified aortic valves. Magnification, 200× and 400×; n = 6. (E) Integrated optical density (IOD)/area of CD11c, CD206 and IL-37. Magnification, 400×, scale bar = 50 μm. (F) CD11c + cell-to-CD206 + cell ratio in CAVD and non-CAVD. Data are presented as the mean ±SD (n = 6); *P < 0.05, **P < 0.01. HE, hematoxylin-eosin; CAVD, calcific aortic valve disease; non-CAVD, non-calcific aortic valve disease.
Fig 3: IL-37 promotes the expression of M2 macrophage markers.(A) Representative immunoblots and densitometric data show that CD206 expression increases after pre-treating PMA-treated THP-1 cells with recombinant human IL-37 (0.1 ng/ml) prior to stimulating with LPS (100 ng/ml) and IFN-? (20 ng/ml); n = 3, *P < 0.05. (B) The ELISA data show that IL-37 up-regulates IL-10 secretion; n = 4, **P < 0.01, ***P < 0.001.
Fig 4: IL-37 modulates macrophage polarization through suppressing activation of the Notch1- NF-?B pathway axis. (A) Representative immunoblots and densitometric data show that stimulation with LPS and IFN-? enhances Notch1 pathway activation in PMA-treated THP-1 cells; n = 3, *P < 0.05, **P < 0.01. (B,C) PMA-treated THP-1 cells were treated with DAPT (50 µM) 1 h prior to LPS (100 ng/ml) and IFN-? (20 ng/ml) for 4–24 h. (B) Representative immunoblots show that DAPT inhibits Notch1 activation; n = 3. (C) Representative immunoblots and densitometric data show that Notch1 pathway inhibition reduces iNOS expression in M1 macrophages; n = 3, *P < 0.05. (D) PMA-treated THP-1 cells were treated with recombinant human IL-37 (0.1 ng/ml) 1 h prior to being treated with LPS (100 ng/ml) and IFN-? (20 ng/ml) at different times. Representative immunoblots and densitometric data show that IL-37 decreases NICD1 expression at different time point in M1 macrophages; n = 3. *P < 0.05 vs. corresponding control, §P < 0.05 vs. M1 macrophages treated with IL-37. (E) Representative immunoblots show that Notch1 pathway inhibition decreases NF-?B phosphorylation; n = 3.
Fig 5: IL-37 suppresses M1 macrophage polarization and pro-inflammatory cytokines secretion. PMA-treated THP-1 cells were treated with LPS (100 ng/ml) and IFN-? (20 ng/ml) in the presence or absence of recombinant IL-37 (0.1 ng/ml). (A,B) Representative immunoblots and densitometric data show that IL-37 inhibits iNOS and CD11c expression; n = 3, *P < 0.05. (C,D) Representative immunoblots and densitometric data show that IL-37 down-regulates MCP-1 and IL-6 expression; n = 3, *P < 0.05. (E,F) The ELISA data show that IL-37 down-regulates MCP-1 and IL-6 secretion; n = 3, *P < 0.05, ***P < 0.001.
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