A rare IL33 loss-of-function mutation reduces blood eosinophil counts and protects from asthma.

Efst á síðunni er hægt að nálgast greinina í heild sinni með því að smella á hlekkinnIL-33 is a tissue-derived cytokine that induces and amplifies eosinophilic inflammation and has emerged as a promising new drug target for asthma and allergic disease. Common variants at IL33 and IL1RL1, encoding the IL-33 receptor ST2, associate with eosinophil counts and asthma. Through whole-genome sequencing and imputation into the Icelandic population, we found a rare variant in IL33 (NM_001199640:exon7:c.487-1G>C (rs146597587-C), allele frequency = 0.65%) that disrupts a canonical splice acceptor site before the last coding exon. It is also found at low frequency in European populations. rs146597587-C associates with lower eosinophil counts (β = -0.21 SD, P = 2.5×10-16, N = 103,104), and reduced risk of asthma in Europeans (OR = 0.47; 95%CI: 0.32, 0.70, P = 1.8×10-4, N cases = 6,465, N controls = 302,977). Heterozygotes have about 40% lower total IL33 mRNA expression than non-carriers and allele-specific analysis based on RNA sequencing and phased genotypes shows that only 20% of the total expression is from the mutated chromosome. In half of those transcripts the mutation causes retention of the last intron, predicted to result in a premature stop codon that leads to truncation of 66 amino acids. The truncated IL-33 has normal intracellular localization but neither binds IL-33R/ST2 nor activates ST2-expressing cells. Together these data demonstrate that rs146597587-C is a loss of function mutation and support the hypothesis that IL-33 haploinsufficiency protects against asthma.Netherlands Asthma Foundation University Medical Center Groningen Ministry of Health and Environmental Hygiene of Netherlands Netherlands Asthma Stichting Astma Bestrijding BBMRI European Respiratory Society private and public research funds AstraZeneca ALK-Abello, Denmar

Nucleotide receptor signalling and the generation of reactive oxygen species

Elevated levels of extracellular nucleotides are present at sites of inflammation, platelet degranulation and cellular damage or lysis. These extracellular nucleotides can lead to the activation of purinergic (nucleotide) receptors on various leukocytes, including monocytes, macrophages, eosinophils, and neutrophils. In turn, nucleotide receptor activation has been linked to increased cellular production and release of multiple inflammatory mediators, including superoxide anion, nitric oxide and other reactive oxygen species (ROS). In the present review, we will summarize the evidence that extracellular nucleotides can facilitate the generation of multiple ROS by leukocytes. In addition, we will discuss several potential mechanisms by which nucleotide-enhanced ROS production may occur. Delineation of these mechanisms is important for understanding the processes associated with nucleotide-induced antimicrobial activities, cell signalling, apoptosis, and pathology

The nucleotide receptor P2RX7 mediates ATP-induced CREB activation in human and murine monocytic cells

Nucleotide receptors serve as sensors of extracellular ATP and are important for immune function. The nucleotide receptor P2RX7 is a cell-surface, ligand-gated cation channel that has been implicated in many diseases, including arthritis, granuloma formation, sepsis, and tuberculosis. These disorders are often exacerbated by excessive mediator release from activated macrophages in the inflammatory microenvironment. Although P2RX7 activation can modulate monocyte/macrophage-induced inflammatory events, the relevant molecular mechanisms are poorly understood. Previous studies suggest that MAPK cascades and transcriptional control via CREB-linked pathways regulate the inflammatory capacity of monocytic cells. As P2RX7 promotes MAPK activation and inflammatory mediator production, we examined the involvement MAPK-induced CREB activation in P2RX7 action. Our data reveal that stimulation of multiple monocytic cell lines with P2RX7 agonists induces rapid CREB phosphorylation. In addition, we observed a lack of nucleotide-induced CREB phosphorylation in RAW 264.7 cells expressing nonfunctional P2RX7 and a gain of nucleotide-induced CREB phosphorylation in human embryonic kidney-293 cells that heterologously express human P2RX7. Furthermore, our results indicate that P2RX7 agonist-induced CREB phosphorylation is partly mediated via Ca2+ fluxes and the MEK/ERK system. Mechanistic analyses revealed that macrophage stimulation with a P2RX7 agonist induces CREB/CREB-binding protein complex formation, which is necessary for CREB transcriptional activation. Also, we demonstrate that P2RX7 activation induces a known CREB-dependent gene (c-fos) and that dominant-negative CREB constructs attenuate this response. These studies support the idea that P2RX7 stimulation can directly regulate protein expression that is not dependent on costimulation with other immune modulators such as LPS

Blood eosinophil counts from wild type and <i>il33</i>-deficient mice.

<p>Blood were collected from male and female 10–12 week old mice and eosinophils were enumerated as described in the online methods. The horizontal bars indicate means within groups. Significance calculated using the unpaired T test, p = 0.014 males, p = 0.0001 females.</p

Conditional analysis for eosinophil counts associations in the region around <i>IL1RL1</i>.

<p>Plot shows an 800kb overview around the <i>IL1RL1</i> gene on chromosome 2. Black circles (o) show-log₁₀ <i>P</i> as a function of hg38 coordinates for unadjusted associations with eosinophil counts; red crosses (+) correspond to eosinophil counts associations after adjusting for the variant rs13020553; blue ‘x’ symbols correspond to eosinophil counts associations after adjusting for both rs13020553 and rs6719123. The position of the two variants rs13020553 and rs6719123 are indicated by vertical broken lines. Genes are shown in blue and recombination rates are reported in cM/Mb.</p

Association of common sequence variants in <i>IL33</i> and <i>IL1RL1</i> with eosinophil counts and asthma in Iceland.

<p>Association of common sequence variants in <i>IL33</i> and <i>IL1RL1</i> with eosinophil counts and asthma in Iceland.</p

RNA analysis of <i>IL33</i> mRNA expression and carrier status of the splice acceptor variant rs146597587.

<p><b>(A)</b> Gene structure of <i>IL33</i> and location of rs146597587 SNP that changes a canonical splice acceptor site from AG to AC. The mutation lies at the junction of coding exons 6 and 7. Exon 7 begins LHKCE and encodes a significant portion of the cytokine domain. Also, indicated is the location of an elastase cleavage site at amino acid 95 that releases the cytokine domain from the N terminus prodomain and leads to a more active cytokine. <b>(B)</b> Expression in FPKM of <i>IL33</i> in adipose tissue for rs146597587 non-carriers (GG, N = 744) and carriers (GC, N = 6) based on RNA-Seq data; <i>P(Wilcox)</i> = 0.0015, median non-carriers: 34.1, carriers: 21.1 (i.e. heterozygotes have 38% lower expression than non-carriers). <b>(C)</b> Ratio of RNA-Seq read coverage in last intron of <i>IL33</i> versus its last exon (splice acceptor resides at this intron-exon boundary) for rs146597587 non-carriers (GG, N = 738) and carriers (GC, N = 9); <i>P</i>(Wilcox) = 2.5×10^–7, mean non-carriers: 0.67%, carriers: 9.0%. <b>(D)</b> Proportion of RNA-Seq read with ALT allele for the synonymous variant rs10975519 (MAF = 29%) for rs146597587 non-carriers (GG, N = 309) and carriers (GC, N = 5); a chromosome carrying rs146597587-C carries the ALT allele of rs10975519; <i>P</i>(Wilcox) = 1.4×10^–4, mean non-carriers: 0.48, non-carriers: 0.20.</p