Hemin and Cobalt Protoporphyrin Inhibit NLRP3 Inflammasome Activation by Enhancing Autophagy: A Novel Mechanism of Inflammasome Regulation

Abstract

Inflammasomes are intracellular protein platforms, which, upon activation, produce the highly proinflammatory cytokines interleukin (IL)-1β and IL-18. Heme, hemin and their degradation products possess significant immunomodulatory functions. Here, we studied whether hemin regulates inflammasome function in macrophages. Both hemin and its derivative, cobalt protoporphyrin (CoPP), significantly reduced IL-1β secretion by cultured human primary macrophages, the human monocytic leukemia cell line and also mouse bone marrow-derived and peritoneal macrophages. Intraperitoneal administration of CoPP to mice prior to urate crystal-induced peritonitis alleviated IL-1β secretion to the peritoneal cavity. In cultured macrophages, hemin and CoPP inhibited NLRP3 inflammasome assembly by reducing the amount of intracellular apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). The reduction of ASC was associated with enhanced autophagosome formation and autophagic flux. Inhibition of autophagy prevented the CoPP-induced depletion of ASC, implying that the depletion was caused by increased autophagy. Our data indicate that hemin functions as an endogenous negative regulator of the NLRP3 inflammasome. The inhibition is mediated via enhanced autophagy that results in increased degradation of ASC. This regulatory mechanism may provide a novel approach for the treatment of inflammasome-related diseases.

© 2016 S. Karger AG, Basel.

Figures

Fig. 1

CoPP and hemin inhibit the secretion of IL-1β by human and mouse macrophages. Unprimed or LPS-primed mouse BMDMs were incubated for 3 h (a, c) or 1 h (b, d) in the presence of CoPP or hemin. The NLRP3 inflammasome was then activated with ATP (a), SAA (b), nigericin (Nig; c) and MSU crystals (d), and the secretion of mature IL-1β was measured by ELISA. Unprimed or LPS-primed human primary macrophages were incubated for 3 h (e) or 1 h (f) in the presence of CoPP or hemin. Thereafter, the stimulation of the NLRP3 inflammasome was induced by ATP (e) or SAA (f). g THP-1 macrophages were incubated for 3 h in the presence of CoPP or hemin, and thereafter activated with nigericin. Secretion of mature IL-1β was measured by ELISA, and is expressed as fold changes compared to the activated cells. Data represent the means ± SD of 3 (c), 4 (a, b, d, f, g) and 6 (e) individual experiments. a–g Statistical significance was evaluated with log2-transformed values. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Fig. 2

CoPP and hemin inhibit inflammasome activation. Cleavage of pro-IL-1β (35 kDa) to mature IL-1β (17 kDa) and of procaspase-1 (45 kDa) to active caspase-1 (20 kDa) were assessed from Western blots of the cell culture media. THP-1 macrophages were incubated for 3 h in the presence of CoPP or hemin (a, c) or for 1 h in the presence of CoPP (b, d), after which, nigericin (Nig; a, c) or SAA (b, d) was added. The Western blots presented are representative of at least 4 individual experiments. e, f Mice were injected i.p. with CoPP (25 mg/kg) or vehicle (PBS) 1 h before challenge with MSU crystals. Peritonitis was allowed to develop for 20 h, after which, the peritoneal cavities of the mice were lavaged. e IL-1β concentration was measured by ELISA and cell numbers (f) were counted from the peritoneal lavage fluid. ** p ≤ 0.01. Each data point in e and f represents 1 mouse; statistical significance was evaluated with log2-transformed values. Data represent the means ± SD.

Fig. 3

Hemin and CoPP do not inhibit priming of NLRP3 inflammasome. a, b Unprimed or LPS-primed human primary macrophages were incubated for 3 h in the presence of CoPP and hemin. LPS-induced expression of IL1B (a) and NLRP3 (b) mRNA was determined by quantitative real-time RT-PCR. The gene expression is expressed as arbitrary units (a.u.). c, d THP-1 macrophages were incubated for 3 h in the presence of CoPP (c) and hemin (d), and activated with nigericin (Nig.). The intracellular levels of NLRP3 (108 kDa), procaspase-1 (45 kDa) and pro-IL-β (37 kDa) were assessed from Western blots of the cell lysates. The Western blots shown are representatives of 4 individual experiments. e To assess the secretion of active IL-18, THP-1 macrophages were incubated for 3 h in the presence of CoPP or hemin, after which, the cells were activated with nigericin. *** p ≤ 0.001; **** p ≤ 0.0001. The secretion of IL-18 was measured by ELISA. Data represent the means ± SD of 3 (e) and 4 (a, b) individual experiments.

Fig. 4

Hemin and CoPP inhibit the release of cathepsin B and the generation of mitochondrial ROS. a THP-1 macrophages were incubated in the presence of CoPP for 3 h and activated with nigericin (Nig.). The potassium level of the cell lysates was determined by ICP-MS. b, c Unprimed or LPS-primed THP-1 macrophages were incubated in the presence of CoPP for 3 h (b) or 1 h (c) before the NLRP3 inflammasome was activated with nigericin (b) or SAA (c), and the release of active lysosomal proteases was detected by measuring the cleavage of cathepsin B/L substrate (excitation at 355 nm, emission at 486 nm) in the cell culture medium. d–f THP-1 macrophages were incubated in the presence of CoPP or hemin for the indicated times and activated with nigericin. d, e The cells were stained with mitoSOX (30 min), detached and analyzed by flow cytometry. Control (Cntr) cells represent the nontreated cells stained with mitoSOX. d The flow cytometric analysis shown is representative of 4 individual experiments. e Fold changes of mean fluorescence of flow cytometric analyses. f THP-1 macrophages were stained with DHR 123, and the fluorescence from cell lysates was analyzed by plate reader (excitation at 500 nm, emission at 536 nm). Given time points indicate the time passed from the administration of CoPP or hemin at the time point of the measurement (see Materials and Methods). Data represent the means ± SD of 3 (a), 4 (e) or 6 (b, c, f) individual experiments. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Fig. 5

Inhibition of HO-1 enzymatic activity reverses the effect of CoPP. The expression of HO-1 protein induced by CoPP or hemin in THP-1 macrophages was determined at 4, 8 and 16 h. a THP-1 macrophages were incubated in the presence of CoPP or hemin for the indicated times, and HO-1 (32 kDa) was assessed from the cell lysates by Western blot (representative of 4 individual experiments). b The enzymatic activity of HO-1 was inhibited by stannous mesoporphyrin (SnMP) for 3 h, prior to the treatment of THP-1 macrophages with CoPP (3 h), after which, the cells were activated with nigericin (Nig). ** p ≤ 0.01. c Unprimed or LPS-primed human primary macrophages were incubated for 1 h in the presence of CORM-3 or its inactivated form (iCORM-3), and thereafter activated with ATP. The secretion of IL-1β was measured by ELISA; statistical significance was evaluated with log2-transformed values. * p ≤ 0.05; ** p ≤ 0.01. Data represent the means ± SD of 3 (b) or 5 (c) individual experiments.

Fig. 6

Hemin and CoPP block ASC speck formation. a, b THP-1 macrophages were incubated for 3 h in the presence of CoPP or hemin, and activated with nigericin (Nig.). Speck formation was visualized by staining for ASC (green) and DNA (blue, DAPI). a Representative of 4 individual experiments. b The extent of speck formation was determined by dividing the number of the specks by the number of nuclei per field. Statistical significance was evaluated with log2-transformed values. Data represent the means ± SD of 4 individual experiments. **** p ≤ 0.0001. c–f THP-1 macrophages were incubated in the presence of CoPP (c, e) or hemin (d, f) and activated with nigericin. Western blots of ASC (22 kDa) (c, d) of the cell lysates and medium (e, f). Western blots shown are representative of 4 (c–e) or 5 (f) individual experiments.

Fig. 7

Induction of autophagy contributes to the degradation of ASC. a, d Unprimed or LPS-primed human primary macrophages were incubated for 3 h in the presence of CoPP or hemin, and then activated with ATP. b, e THP-1 macrophages were incubated for 3 h in the presence of CoPP or hemin, and thereafter activated with nigericin (Nig.). a Western blot analysis and quantitation of LC3 (LC3-I 19 kDa and LC3-II 17 kDa) bands of human primary macrophage lysates. b Western blot analysis and quantitation of LC3 (LC3-I 19 kDa and LC3-II 17 kDa) bands of THP-1 macrophage lysates. ** p ≤ 0.01. a, b In the quantitations, the ratio of the intensities of the bands of LC3-I and LC3-II (LC3-II/LC3-I) are expressed as fold changes compared to the LC3 ratio determined from the nontreated cells. Data represent the means ± SD. c THP-1 macrophages were incubated in the presence of CoPP for the indicated times, and stained for SQSTM1 (green), ASC (red) and DNA (blue, DAPI; colors refer to the online version). d Western blot analysis of SQSTM1 (62 kDa) of human primary macrophage lysates. e Western blot analysis of SQSTM1 (62 kDa) of THP-1 macrophage lysates. f THP-1 macrophages were first incubated in the presence of autophagy inhibitor 3-MA, then for 3 h in the presence of CoPP, and finally activated with nigericin. Intracellular ASC (22 kDa) was assessed from the cell lysates by Western blot analysis. The Western blots or immunofluorescence microscopy images shown are representative of 3 (b), 4 (a, c, f) or 5 (d, e) individual experiments.