Native and oxidised lipoproteins negatively regulate the serum amyloid A-induced NLRP3 inflammasome activation in human macrophages.

OBJECTIVES: The NLRP3 inflammasome plays a key role in arterial wall inflammation. In this study, we elucidated the role of serum lipoproteins in the regulation of NLRP3 inflammasome activation by serum amyloid A (SAA) and other inflammasome activators. METHODS: The effect of lipoproteins on the NLRP3 inflammasome activation was studied in primary human macrophages and THP-1 macrophages. The effect of oxidised low-density lipoprotein (LDL) was examined in an in vivo mouse model of SAA-induced peritoneal inflammation. RESULTS: Native and oxidised high-density lipoproteins (HDL3) and LDLs inhibited the interaction of SAA with TLR4. HDL3 and LDL inhibited the secretion of interleukin (IL)-1ß and tumor necrosis factor by reducing their transcription. Oxidised forms of these lipoproteins reduced the secretion of mature IL-1ß also by inhibiting the activation of NLRP3 inflammasome induced by SAA, ATP, nigericin and monosodium urate crystals. Specifically, oxidised LDL was found to inhibit the inflammasome complex formation. No cellular uptake of lipoproteins was required, nor intact lipoprotein particles for the inhibitory effect, as the lipid fraction of oxidised LDL was sufficient. The inhibition of NLRP3 inflammasome activation by oxidised LDL was partially dependent on autophagy. Finally, oxidised LDL inhibited the SAA-induced peritoneal inflammation and IL-1ß secretion in vivo. CONCLUSIONS: These findings reveal that both HDL3 and LDL inhibit the proinflammatory activity of SAA and this inhibition is further enhanced by lipoprotein oxidation. Thus, lipoproteins possess major anti-inflammatory functions that hinder the NLRP3 inflammasome-activating signals, particularly those exerted by SAA, which has important implications in the pathogenesis of cardiovascular diseases.

Ethanol inhibits activation of NLRP3 and AIM2 inflammasomes in human macrophages--a novel anti-inflammatory action of alcohol.

OBJECTIVE: In the pathogenesis of coronary atherosclerosis, local macrophage-driven inflammation and secretion of proinflammatory cytokines, interleukin-1ß (IL-1ß) in particular, are recognized as key factors. Moderate alcohol consumption is associated with a reduced risk of coronary artery disease mortality. Here we examined in cultured human macrophages whether ethanol modulates the intracellular processes involved in the secretion of IL-1ß. RESULTS: Ethanol decreased dose-dependently the production of mature IL-1ß induced by activators of the NLRP3 inflammasome, i.e. ATP, cholesterol crystals, serum amyloid A and nigericin. Ethanol had no significant effect on the expression of NLRP3 or IL1B mRNA in LPS-primed macrophages. Moreover, secretion of IL-1ß was decreased in parallel with reduction of caspase-1 activation, demonstrating that ethanol inhibits inflammasome activation instead of synthesis of pro-IL-1ß. Acetaldehyde, a highly reactive metabolite of ethanol, had no effect on the ATP-induced IL-1ß secretion. Ethanol also attenuated the secretion of IL-1ß triggered by synthetic double-stranded DNA, an activator of the AIM2 inflammasome. Ethanol conferred the inhibitory functions by attenuating the disruption of lysosomal integrity and ensuing leakage of the lysosomal protease cathepsin B and by reducing oligomerization of ASC. CONCLUSION: Ethanol-induced inhibition of the NLRP3 inflammasome activation in macrophages may represent a biological pathway underlying the protective effect of moderate alcohol consumption on coronary heart disease.

Serum amyloid A activates the NLRP3 inflammasome via P2X7 receptor and a cathepsin B-sensitive pathway.

Serum amyloid A (SAA) is an acute-phase protein, the serum levels of which can increase up to 1000-fold during inflammation. SAA has a pathogenic role in amyloid A-type amyloidosis, and increased serum levels of SAA correlate with the risk for cardiovascular diseases. IL-1ß is a key proinflammatory cytokine, and its secretion is strictly controlled by the inflammasomes. We studied the role of SAA in the regulation of IL-1ß production and activation of the inflammasome cascade in human and mouse macrophages, as well as in THP-1 cells. SAA could provide a signal for the induction of pro-IL-1ß expression and for inflammasome activation, resulting in secretion of mature IL-1ß. Blocking TLR2 and TLR4 attenuated SAA-induced expression of IL1B, whereas inhibition of caspase-1 and the ATP receptor P2X(7) abrogated the release of mature IL-1ß. NLRP3 inflammasome consists of the NLRP3 receptor and the adaptor protein apoptosis-associated speck-like protein containing CARD (a caspase-recruitment domain) (ASC). SAA-mediated IL-1ß secretion was markedly reduced in ASC(-/-) macrophages, and silencing NLRP3 decreased IL-1ß secretion, confirming NLRP3 as the SAA-responsive inflammasome. Inflammasome activation was dependent on cathepsin B activity, but it was not associated with lysosomal destabilization. SAA also induced secretion of cathepsin B and ASC. In conclusion, SAA can induce the expression of pro-IL-1ß and activation of the NLRP3 inflammasome via P2X(7) receptor and a cathepsin B-sensitive pathway. Thus, during systemic inflammation, SAA may promote the production of IL-1ß in tissues. Furthermore, the SAA-induced secretion of active cathepsin B may lead to extracellular processing of SAA and, thus, potentially to the development of amyloid A amyloidosis.

Serum amyloid A (SAA) activates human mast cells which leads into degradation of SAA and generation of an amyloidogenic SAA fragment.

Serum amyloid A (SAA) is a precursor for the amyloid A in AA type of amyloidosis. Distribution of mast cells in tissues is similar to the distribution of amyloid deposits in secondary AA-amyloidosis. Therefore, we studied whether mast cells could be involved in SAA metabolism. Human mast cell line (HMC-1) cells were cultured with recombinant human apoSAA (rhSAA), and the production of tumour necrosis factor (TNF)-alpha and interleukin (IL)-1 beta was determined by ELISA. RhSAA and human SAA (huSAA) were incubated with human chymase, tryptase or with intact human mast cell (huMC) in cultures, and degradation of SAA was followed by gel electrophoresis, liquid chromatography and mass spectrometry. SAA induced dose-dependent production of TNF-alpha and IL-1 beta in HMC-1 cells. Tryptase, chymase, and huMC granules degraded efficiently the SAA protein. Degradation of SAA by tryptase, but not by chymase, released a highly amyloidogenic N-terminal fragment of SAA. Finally, incubation of huMC with rhSAA alone resulted in degradation of SAA and formation of protofibrillar intermediates. These results suggest a pathogenic role for mast cells in AA-amyloidosis.

Selective binding of synapse-associated protein 97 to GluR-A alpha-amino-5-hydroxy-3-methyl-4-isoxazole propionate receptor subunit is determined by a novel sequence motif.

A family of four closely related PDZ domain-containing membrane-associated guanylate kinase homologues (MAGUKs) is involved in the regulation of the amount and functional state of ionotropic glutamate receptors in excitatory synapses. To understand the mechanisms that determine the specificity of these interactions, we examined the structural basis of the highly selective association between the ionotropic GluR subunit GluR-A and synapse-associated protein 97 (SAP97). The C terminus of GluR-A bound to the PDZ domains of SAP97, but not to those of three related MAGUKs, PSD-93, PSD-95, and SAP102. Experiments with single PDZ domains indicated that the strongest contribution was by the second PDZ domain. Unexpectedly, mutation analysis of the GluR-A C terminus revealed that a tripeptide sequence SSG at position -9 to -11 plays an essential role in this binding, in addition to a C-terminal type I PDZ binding motif (leucine at C terminus and threonine at the -2 position). Analysis of the in vitro MAGUK-binding properties of a GluR-D mutant with a one-residue deletion at the C terminus provides further support for the view that an SSG sequence located N-terminally from a type I PDZ binding motif can mediate selective binding to SAP97 and suggest the existence of a novel variation of the PDZ domain-peptide interaction.