Kristen K. Skarratt

A thr357 to ser polymorphism in homozygous and compound heterozygous subjects causes absent or reduced P2X7 function and impairs atp-induced mycobacterial killing by macrophages

The P2X7 receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X7 receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5′-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X7 polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C → G), which changes Thr357 to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X7 function was measured by ATP-induced ethidium+ influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10–65% in heterozygotes, 1–18% in homozygotes, and 0–10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X7 in either HEK-293 cells or Xenopus oocytes gave P2X7 function of ∼50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X7, restored P2X7 function to near normal in cells heterozygous for T357S and to a value 50–65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X7 function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X7 receptor function.

Two haplotypes of the P2X7 receptor containing the Ala-348 to Thr polymorphism exhibit a gain-of-function effect and enhanced interleukin-1β secretion

The P2X(7) receptor is an ATP-gated cation channel expressed in immune cells and plays a role in proinflammatory cytokine release from monocytes and macrophages. This study investigated the coinheritance of 12 functionally relevant single nucleotide polymorphisms (SNPs) in the human P2X(7) gene (P2RX7), and the functional effect of each singly and in combination was assessed by measurements of ATP-induced currents and ethidium(+) uptake. Genotyping of 3430 Caucasian subjects identified 4 common haplotypes in addition to the common (wild-type) P2X(7)-1. Two haplotypes (denoted P2X(7)-2 and P2X(7)-4) contained various combinations of gain-of-function SNPs. P2X(7)-4 was identified uniquely by the Gln-460 to Arg polymorphism (rs2230912). When expressed in HEK-293 cells, recombinant P2X(7)-2, and P2X(7)-4 haplotypes displayed a 3-fold and 5-fold increase, respectively, in receptor function compared to the wild-type P2X(7)-1. Both P2X(7) haplotypes contained the Ala-348>Thr polymorphism (rs1718119), and this mutation was critical for the gain-of-function effect. Peripheral blood monocytes and erythrocytes from subjects homozygous for gain-of-function P2X(7) haplotypes exhibited increased ATP-induced ethidium(+) uptake and (86)Rb(+) efflux, respectively, and this correlated with increased IL-1beta secretion from LPS-primed monocytes. Inheritance of these P2X(7) haplotypes predisposing to increased proinflammatory cytokine secretion may be important in genetic association studies of inflammatory, infectious, and psychiatric disorders.

Genetics of the P2X7 receptor and human disease

The P2RX7 gene is highly polymorphic, and many single nucleotide polymorphisms (SNPs) underlie the wide variation observed in P2X7 receptor responses. We review the discovery of those non-synonymous SNPs that affect receptor function and compare their frequencies in different ethnic populations. Analysis of pairwise linkage disequilibrium (LD) predicts a limited range of haplotypes. The strong LD between certain functional SNPs provides insight into published studies of the association between SNPs and human disease.

Gene Dosage Determines the Negative Effects of Polymorphic Alleles of the P2X7 Receptor on Adenosine Triphosphate–Mediated Killing of Mycobacteria by Human Macrophages

BACKGROUND Stimulation of the P2X7 purinergic receptor (P2X7) in bacille Calmette-Guerin (BCG)-infected human macrophages with extracellular adenosine triphosphate (ATP) leads to pore formation and killing of mycobacteria. We examined the effect of polymorphisms in the P2X7 gene (P2X7) on the capacity of macrophages to kill mycobacteria. METHODS Polymorphisms and mutations in P2X7 were identified by both DNA sequence analysis and determination of uptake of ethidium by time-resolved flow cytometry. Macrophages from affected subjects were infected with Mycobacterium bovis BCG. Apoptosis was determined by use of Annexin V staining, and BCG growth was determined by use of quantitative mycobacterial cultures. RESULTS Three new mutations were identified. Macrophages from subjects heterozygous for a polymorphism in P2X7 had a 50% reduction in uptake of ethidium and a 75% reduction in the number of apoptotic cells, compared with macrophages from wild-type (wt) subjects, after stimulation with interferon (IFN)- gamma and ATP. Furthermore, after stimulation with IFN- gamma and ATP, there was a reduction in BCG growth of up to approximately 0.5 log10 in macrophages from single-heterozygous subjects, compared with a reduction of 1.0 log10 in macrophages from wt subjects. Interestingly, BCG-infected macrophages from compound-heterozygous subjects, for different combinations of polymorphisms in P2X7, had no uptake of ethidium, failed to undergo apoptosis, and were unable to kill mycobacteria after stimulation with IFN- gamma and ATP. CONCLUSIONS Various polymorphisms in P2X7 abrogate IFN- gamma /ATP-induced killing of mycobacteria by human macrophages and, thus, may contribute to variability in susceptibility to mycobacterial infections.

A 5′ intronic splice site polymorphism leads to a null allele of the P2X7 gene in 1–2% of the Caucasian population

The P2X7 gene is important for the innate immune response but known polymorphisms do not explain all subjects with loss of P2X7 function. A splice site mutation (g → t) was found at position +1 of the first intron of the P2X7 gene in 7 of 336 Caucasians and 1 of 39 subjects of Indian ethnicity. All eight subjects were heterozygous for the uncommon 1513A → C polymorphism of the P2X7 gene. RT‐PCR and sequencing showed the splice site mutation was on the 1513C allele in the Caucasians and on the 1513A allele in the Indian subject. The splice site mutation is an inherited polymorphism and gives rise to a P2X7 null allele in 1–2% of the Caucasian population.

Single-nucleotide polymorphisms in the P2X7 receptor gene are associated with post-menopausal bone loss and vertebral fractures

The purinergic P2X7 receptor has a major role in the regulation of osteoblast and osteoclast activity and changes in receptor function may therefore affect bone mass in vivo. The aim of this study was to determine the association of non-synonymous single-nucleotide polymorphisms in the P2RX7 gene to bone mass and fracture incidence in post-menopausal women. A total of 1694 women (aged 45–58) participating in the Danish Osteoporosis Prevention Study were genotyped for 12 functional P2X7 receptor variants. Bone mineral density was determined at baseline and after 10 years. In addition, vertebral fracture incidence was documented at 10 years. We found that the rate of bone loss was clearly associated with the Arg307Gln amino acid substitution such that individuals heterozygous for this polymorphism had a 40% increased rate of bone loss. Furthermore, individuals carrying the Ile568Asn variant allele had increased bone loss. In contrast, the Gln460Arg polymorphism was associated with protection against bone loss. The Ala348Thr polymorphism was associated with a lower vertebral fracture incidence 10 years after menopause. Finally, we developed a risk model, which integrated P2RX7 genotypes. Using this model, we found a clear association between the low-risk (high-P2X7 function) alleles and low rate of bone loss. Conversely, high-risk (reduced P2X7 function) alleles were associated with a high rate of bone loss. In conclusion, an association was demonstrated between variants that reduce P2X7 receptor function and increased rate of bone loss. These data support that the P2X7 receptor is important in regulation of bone mass.

Polymorphisms in the P2X7 receptor gene are associated with low lumbar spine bone mineral density and accelerated bone loss in post-menopausal women

The P2X7 receptor gene (P2RX7) is highly polymorphic with five previously described loss-of-function (LOF) single-nucleotide polymorphisms (SNP; c.151+1G>T, c.946G>A, c.1096C>G, c.1513A>C and c.1729T>A) and one gain-of-function SNP (c.489C>T). The purpose of this study was to determine whether the functional P2RX7 SNPs are associated with lumbar spine (LS) bone mineral density (BMD), a key determinant of vertebral fracture risk, in post-menopausal women. We genotyped 506 post-menopausal women from the Aberdeen Prospective Osteoporosis Screening Study (APOSS) for the above SNPs. Lumbar spine BMD was measured at baseline and at 6–7 year follow-up. P2RX7 genotyping was performed by homogeneous mass extension. We found association of c.946A (p.Arg307Gln) with lower LS-BMD at baseline (P=0.004, β=−0.12) and follow-up (P=0.002, β=−0.13). Further analysis showed that a combined group of subjects who had LOF SNPs (n=48) had nearly ninefold greater annualised percent change in LS-BMD than subjects who were wild type at the six SNP positions (n=84; rate of loss=−0.94%/year and −0.11%/year, respectively, P=0.0005, unpaired t-test). This is the first report that describes association of the c.946A (p.Arg307Gln) LOF SNP with low LS-BMD, and that other LOF SNPs, which result in reduced or no function of the P2X7 receptor, may contribute to accelerated bone loss. Certain polymorphic variants of P2RX7 may identify women at greater risk of developing osteoporosis.

IL-10 Potentiates Differentiation of Human Induced Regulatory T Cells via STAT3 and Foxo1

Foxp3+ regulatory T cells (Tregs) play essential roles in maintaining the immune balance. Although the majority of Tregs are formed in the thymus, increasing evidence suggests that induced Tregs (iTregs) may be generated in the periphery from naive cells. However, unlike in the murine system, significant controversy exists regarding the suppressive capacity of these iTregs in humans, especially those generated in vitro in the presence of TGF-β. Although it is well known that IL-10 is an important mediator of Treg suppression, the action of IL-10 on Tregs themselves is less well characterized. In this article, we show that the presence of IL-10, in addition to TGF-β, leads to increased expansion of Foxp3+ iTregs with enhanced CTLA-4 expression and suppressive capability, comparable to that of natural Tregs. This process is dependent on IL-10R–mediated STAT3 signaling, as supported by the lack of an IL-10 effect in patients with IL-10R deficiency and dominant-negative STAT3 mutation. Additionally, IL-10–induced inhibition of Akt phosphorylation and subsequent preservation of Foxo1 function are critical. These results highlight a previously unrecognized function of IL-10 in human iTreg generation, with potential therapeutic implications for the treatment of immune diseases, such as autoimmunity and allergy.

Probenecid Blocks Human P2X7 Receptor-Induced Dye Uptake via a Pannexin-1 Independent Mechanism

P2X7 is a ligand-gated ion channel which is activated by ATP and displays secondary permeability characteristics. The mechanism of development of the secondary permeability pathway is currently unclear, although a role for the hemichannel protein pannexin-1 has been suggested. In this study we investigated the role of pannexin-1 in P2X7-induced dye uptake and ATP-induced IL-1β secretion from human monocytes. We found no pharmacological evidence for involvement of pannexin-1 in P2X7-mediated dye uptake in transfected HEK-293 cells with no inhibition seen for carbenoxolone and the pannexin-1 mimetic inhibitory peptide, 10Panx1. However, we found that probenecid inhibited P2X7-induced cationic and anionic dye uptake in stably transfected human P2X7 HEK-293 cells. An IC50 value of 203 μM was calculated for blockade of ATP-induced responses at human P2X7. Probenecid also reduced dye uptake and IL-1β secretion from human CD14+ monocytes whereas carbenoxolone and 10Panx1 showed no inhibitory effect. Patch clamp and calcium indicator experiments revealed that probenecid directly blocks the human P2X7 receptor.

Association of the 1513C polymorphism in the P2X7 gene with familial forms of chronic lymphocytic leukaemia

Association of the 1513C polymorphism in the P2X7 gene with familial forms of chronic lymphocytic leukaemia.