DISSECTING THE MECHANISM OF NLRP3 INFLAMMASOME ACTIVATION IN INDIVIDUAL CELLS: THE ROLE OF REACTIVE OXYGEN SPECIES AND ORGANELLE DAMAGE

Inflammation underlies the pathology of numerous diseases. It can be initiated by macrophages through the secretion of pro-inflammatory cytokines, such as IL-18 and IL-1β, following the activation of a molecular complex called the inflammasome. Inflammasomes are a protein scaffolding complex consisting of three known components: a sensory NLR, such as NLRP3, ASC, and caspase-1. The NLRP3 inflammasome is activated by a diverse array of stimuli, including crystals, ATP, pore-forming toxins, such as tetanolysin O (TLO), and the potassium ionophore nigericin. Here, we have explored the mechanism of NLRP3 inflammasome activation using nigericin, TLO and ATP. We found that nigericin induced NLRP3 inflammasome activation in bone-marrow derived macrophages (BMDM) results in inflammasome dependent lysosomal membrane permeabilization (LMP), mitochondrial membrane permeabilization (MMP), and the processing and secretion of IL-1β. All of these events required mitochondrial reactive oxygen species (ROS). Through combining bulk biochemical assays with live cell analysis of individual cells, we provide a kinetic analysis and sequence of events resulting from nigericin stimulation of LPS primed BMDM. We recapitulated NLRP3 inflammasome activation in a dendritic cell line, using the novel system of D2SC-1 transduced with ASC. These transduced cells undergo a similar sequence of events as macrophages, confirming that this mechanism is a general result of NLRP3 inflammasome activation applicable to multiple cell types. Furthermore, we have found that ATP and TLO stimulation of BMDM results in a similar sequence of NLRP3 dependent events. ATP, but not nigericin, requires P2X7 for activation of the NLRP3 inflammasome. Surprisingly, inhibition of P2X4 blocked nigericin, but not ATP, induced, NLRP3-dependent IL-1β secretion in BMDM. Our work has demonstrated a central, common role for mitochondrial ROS in NLRP3 inflammasome activation and determined the kinetics of organelle crosstalk during inflammasome activation. These data place both the mitochondria and lysosomes in a critical position controlling NLRP3 inflammasome activation. Based on these results, we suggest mitochondrial ROS as a potential therapeutic target for treating NLRP3 inflammasome related diseases. Inhibition or scavenging of mitochondrial ROS would not only prevent the pro-inflammatory effects of IL-1β secretion in these patients, but also NLRP3 dependent organelle damage and the resulting cell death

The Second Transmembrane Domain of P2X7 Contributes to Dilated Pore Formation

Activation of the purinergic receptor P2X7 leads to the cellular permeability of low molecular weight cations. To determine which domains of P2X7 are necessary for this permeability, we exchanged either the C-terminus or portions of the second transmembrane domain (TM2) with those in P2X1 or P2X4. Replacement of the C-terminus of P2X7 with either P2X1 or P2X4 prevented surface expression of the chimeric receptor. Similarly, chimeric P2X7 containing TM2 from P2X1 or P2X4 had reduced surface expression and no permeability to cationic dyes. Exchanging the N-terminal 10 residues or C-terminal 14 residues of the P2X7 TM2 with the corresponding region of P2X1 TM2 partially restored surface expression and limited pore permeability. To further probe TM2 structure, we replaced single residues in P2X7 TM2 with those in P2X1 or P2X4. We identified multiple substitutions that drastically changed pore permeability without altering surface expression. Three substitutions (Q332P, Y336T, and Y343L) individually reduced pore formation as indicated by decreased dye uptake and also reduced membrane blebbing in response to ATP exposure. Three others substitutions, V335T, S342G, and S342A each enhanced dye uptake, membrane blebbing and cell death. Our results demonstrate a critical role for the TM2 domain of P2X7 in receptor function, and provide a structural basis for differences between purinergic receptors. © 2013 Sun et al

A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants.

This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ng.3448Advanced age-related macular degeneration (AMD) is the leading cause of blindness in the elderly, with limited therapeutic options. Here we report on a study of >12 million variants, including 163,714 directly genotyped, mostly rare, protein-altering variants. Analyzing 16,144 patients and 17,832 controls, we identify 52 independently associated common and rare variants (P < 5 × 10(-8)) distributed across 34 loci. Although wet and dry AMD subtypes exhibit predominantly shared genetics, we identify the first genetic association signal specific to wet AMD, near MMP9 (difference P value = 4.1 × 10(-10)). Very rare coding variants (frequency <0.1%) in CFH, CFI and TIMP3 suggest causal roles for these genes, as does a splice variant in SLC16A8. Our results support the hypothesis that rare coding variants can pinpoint causal genes within known genetic loci and illustrate that applying the approach systematically to detect new loci requires extremely large sample sizes.We thank all participants of all the studies included for enabling this research by their participation in these studies. Computer resources for this project have been provided by the high-performance computing centers of the University of Michigan and the University of Regensburg. Group-specific acknowledgments can be found in the Supplementary Note. The Center for Inherited Diseases Research (CIDR) Program contract number is HHSN268201200008I. This and the main consortium work were predominantly funded by 1X01HG006934-01 to G.R.A. and R01 EY022310 to J.L.H

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Genetic loci and prioritization of genes for kidney function decline derived from a meta-analysis of 62 longitudinal genome-wide association studies

Estimated glomerular filtration rate (eGFR) reflects kidney function. Progressive eGFR-decline can lead to kidney failure, necessitating dialysis or transplantation. Hundreds of loci from genome-wide association studies (GWAS) for eGFR help explain population cross section variability. Since the contribution of these or other loci to eGFR-decline remains largely unknown, we derived GWAS for annual eGFR-decline and meta-analyzed 62 longitudinal studies with eGFR assessed twice over time in all 343,339 individuals and in high-risk groups. We also explored different covariate adjustment. Twelve genome-wide significant independent variants for eGFR-decline unadjusted or adjusted for eGFR-baseline (11 novel, one known for this phenotype), including nine variants robustly associated across models were identified. All loci for eGFR-decline were known for cross-sectional eGFR and thus distinguished a subgroup of eGFR loci. Seven of the nine variants showed variant-by-age interaction on eGFR cross section (further about 350,000 individuals), which linked genetic associations for eGFR-decline with age-dependency of genetic cross-section associations. Clinically important were two to four-fold greater genetic effects on eGFR-decline in high-risk subgroups. Five variants associated also with chronic kidney disease progression mapped to genes with functional in-silico evidence (UMOD, SPATA7, GALNTL5, TPPP). An unfavorable versus favorable nine-variant genetic profile showed increased risk odds ratios of 1.35 for kidney failure (95% confidence intervals 1.03-1.77) and 1.27 for acute kidney injury (95% confidence intervals 1.08-1.50) in over 2000 cases each, with matched controls). Thus, we provide a large data resource, genetic loci, and prioritized genes for kidney function decline, which help inform drug development pipelines revealing important insights into the age-dependency of kidney function genetics

Point mutations of P2X7 TM2 alter cell viability.

<p>NRK cells stably transduced with the indicated constructs were imaged at 2 frames/minute for 45 minutes in the presence of DAPI (blue) and PI (red) following the addition of 3 mM ATP. Arrowheads indicate blebs. Images shown are representative of at least 10 fields of cells from at least two independent experiments. Scale bar = 20 µm.</p

The TM2 of P2X7 confers surface expression and channel activity.

<p>(A) Schematic representation of the chimeric constructs used. All constructs are P2X7 with either the entire TM2 replaced with that of P2X1 (TM21), that of P2X4 (TM24) or the first or second half of TM2 replaced with that of P2X1 (TM21N and TM21C, respectively). A sequence alignment of P2X1, P2X4 and P2X7 are shown with *, :and. indicating identical, conserved and similar residues, respectively. (B, C) HEK293 cells stably expressing the vector alone (mock), P2X7 or the chimeric constructs were either (B) surface stained with anti-P2X7 mAb HANO43 or (C) treated with 3 mM ATP for 30 min in the presence of DAPI, Etd, YoPro1 and PI and analyzed by flow cytometry. The data shown are representative of more than three independent experiments and two tailed Student's t-tests (unpaired) compared to mock yielded p values of <0.05 (*), <0.01 (**), or <0.001(***) while comparisons to wild type P2X7 yielded p values <0.05 (#).</p

P2X7 mediates rapid ATP-dependent DAPI uptake.

<p>(A) NRK cell lysates (30 µg/lane) stably expressing P2X1, P2X4, P2X7 or vector alone (mock) were resolved by SDS-PAGE, transferred to PVDF and membranes probed with the indicated antibodies. (B) NRK cells stably expressing P2X7 were treated with the indicated concentrations ATP for 15 min and DAPI uptake analyzed by flow cytometry. (C) NRK cells stably expressing either P2X7 or vector alone (mock) were incubated with the indicated concentrations of ATP for the indicated times, and DAPI uptake measured by flow cytometry. The geometric mean fluorescence intensity (GMFI) is shown. (D, E) NRK cells stably expressing P2X1, P2X4, P2X7 or vector alone (mock) were treated with ATP for 15 min and uptake of DAPI (D), Etd, YoPro1, and PI (E) measured by flow cytometry. Fold fluorescent increase was determined by dividing the GMFI of cells in the presence of ATP by the GMFI in the same cells without addition of ATP (GMFI_ATP/GMFI_no-ATP). (F) NRK cells stably expressing P2X1, P2X4, P2X7 or vector alone (mock) were treated with ATP for 15 min in the absence (NT) or presence of 100 µM A74003 (A74) or 10 µM A438079 (A43) and uptake of DAPI was measured by flow cytometry. The data shown are representative of more than three independent experiments and two tailed Student's t-tests (unpaired) compared to mock yielded p values <0.05 (*), <0.01 (**), or <0.001(***).</p