Novel NLRP3/cryopyrin mutations and pro-inflammatory cytokine profiles in Behçet's syndrome patients

The role of mutations in NLRP3 in inflammatory features of Behçet's syndrom

Artificial Loading of ASC Specks with Cytosolic Antigens

<div><p>Inflammasome complexes form upon interaction of Nod Like Receptor (NLR) proteins with pathogen associated molecular patterns (PAPMS) inside the cytosol. Stimulation of a subset of inflammasome receptors including NLRP3, NLRC4 and AIM2 triggers formation of the micrometer-sized spherical supramolecular complex called the ASC speck. The ASC speck is thought to be the platform of inflammasome activity, but the reason why a supramolecular complex is preferred against oligomeric platforms remains elusive. We observed that a set of cytosolic proteins, including the model antigen ovalbumin, tend to co-aggregate on the ASC speck. We suggest that co-aggregation of antigenic proteins on the ASC speck during intracellular infection might be instrumental in antigen presentation.</p></div

ASC specks stably co-aggregate some but not all cytosolic proteins.

<p>Fluorescently tagged (mCherry) ASC protein was co-expressed with a set of EGFP-tagged constructs in HEK293T cells. Representative constructs: (A) EGFP-C3 and EGFP alone (B) cOVA-EYFP (1-48aa secretion signal deleted) and EYFP alone were co-expressed with mCherry-ASC. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134912#pone.0134912.s001" target="_blank">S1 Fig</a> for other constructs tested. (C) Shorter versions of C3 and peptide 1 were cloned to C-terminus of EGFP. Only EGFP-peptide 1_19aa co-aggregated on ASC specks, but EGFP-C3_19aa, -peptide 1_12aa and-peptide 1_8aa did not. (D) 2 hydrophobic and 2 hydrophilic randomly generated peptide encoding sequences were cloned to C-terminus of EGFP. EGFP-hydrophobic peptides but not EGFP-hydrophilic peptides co-aggregated on ASC specks. Results are representative of two independent experiments. (E) Hydropathy plots of peptides co-aggregating (peptide 1 and C3) or not co-aggregating (peptide 2 and 3) on ASC specks when fused with EGFP and co-expressed with mCherry-ASC construct. Y-axis: Hydrophobicity values according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134912#pone.0134912.ref038" target="_blank">38</a>]. X-axis: Amino acid position. Columns above the x-axis correspond to hydrophilic peptides and below the x-axis correspond to hydrophobic peptides. (F) EGFP-C3 co-aggregated ASC specks were extracted from HEK293T cells. Samples were imaged right after extraction and after incubation at 37°C for 30 days in PBS. Results are representative of at least two independent experiments. Scale bar: 10 μm.</p

Co-localization frequency of ovalbumin on NLRP3-induced ASC specks is increased by proteasomal inhibition.

<p>HEK293T cells were transduced with lentiviral particles to stably express mCherry-ASC fusion protein. Inverted fluorescent microscopy images of cells, transfected either with empty (pcDNA3) or NLRP3-encoding plasmids, to induce ASC speck formation. Cells were also co-transfected with either (A) EYFP alone (control) or (B) cOVA-EYFP Scale bar (A) and (B): 100 μm. 24 h after transfection, cells were treated with either mock or 10 μM MG132 for 6 h. Exposure time of EYFP alone images is kept 4x shorter than cOVA-EYFP to avoid saturation of pixels due to fluorescence intensity differences. Close-up confocal micrographs of (C) EYFP or (D) cOVA-EYFP expressing NLRP3-induced ASC speck forming cells. Scale bar (C) and (D): 10 μm (E) Western blotting analyses of samples in A-B. EYFP and cOVA-EYFP were exposed equally (marked with asterix). cOVA-EYFP was also exposed longer due to low intensity of the bands. (F) Co-localization frequency of ovalbumin on NLRP3-induced ASC specks either in the absence or presence of MG132 (p < 0.0001, n = 4). (G) NLRP3-induced ASC specks per visual field either in the absence or presence of MG132 (not significant, p = 0.48, n = 4). Results are representative of two independent experiments.</p

Extracellular ASC specks were released from THP-1 macrophages treated with MSU crystals, and can be engulfed and processed by another macrophage.

<p>(A) Human monocytic THP-1 cells were transduced with lentiviral particles to stably express EGFP-ASC fusion protein. THP-1 cells were differentiated with PMA. Differentiated cells were treated with 150 μg/ml MSU crystals for 24 h. ASC speck were observed in the extracellular space (marked with arrows). Bright field is converted into red channel in the overlay image for better visual seperation. Scale bar (A): 100 μm. (B) EGFP-tagged ASC specks were purified from HEK293T cells and incubated with PMA-differentiated THP-1 cells for 3 h. Presence of engulfed ASC speck in an acidic organelle in THP-1 macrophage was demonstrated by Lysotracker Red staining. (C) Time-lapse imaging of PMA-differentiated stably EGFP-ASC expressing THP-1 macrophages that engulfed extracellular mCherry-tagged ASC specks. Tubular vesicles trafficking from the phagolysosome which contained engulfed ASC speck were observed. Cytosolic stable EGFP-ASC expression (diffused green signal) and engulfed mCherry-ASC speck were observed as two distinct compartments within the same cell. Time-lapse imaging was carried out using an in-house produced growth chamber. Results are representative of at least two independent experiments. Scale bar (B) and (C): 25 μm.</p

Novel NLRP3/cryopyrin mutations and pro-inflammatory cytokine profiles in Behcet's syndrome patients

Behçet's syndrome (BS) is a systemic inflammatory disorder with unknown etiology. Features of both innate and adaptive immunity have been claimed in the pathogenesis of BS. To test the possible dysregulation of the NLRP3/cryopyrin (Nod-like receptor with a pyrin domain 3) inflammasome, as a result of mutation(s), we performed single-strand conformation polymorphism analyses and/or sequencing of all the coding regions and intron-exon boundaries of NLRP3/cryopyrin and ASC (apoptosis-associated speck-like protein containing CARD) genes from Turkish BS patients and healthy controls. At the same time, we determined pro-inflammatory cytokine secretion profiles of peripheral blood cells in response to LPS treatment using ELISA. BS patients with vascular involvement showed significantly increased levels of TNF-α release at 2-, 4- and 8-h post-treatment and significantly increased IL-1β levels were detected at 2h (P = 0.005) and 4h (P = 0.025) (n = 10). We identified four mutations in the NLRP3/cryopyrin gene, V200M (n = 3/104) and T195M (n = 1/104), in BS patients but none in control samples. No mutations were detected in the ASC gene. The effect of these NLRP3/cryopyrin mutants on ASC speck assembly and IL-1β secretion was tested and the V200M mutant was shown to induce IL-1β secretion. Thus, it is likely that certain mutations in NLRP3/cryopyrin in combination with yet unknown other factors may contribute to the pro-inflammatory cytokine profiles in BS patients