The Presence of P2RX7 Single Nuclear Polymorphism Is Associated

The presence of P2RX7 single nuclear polymorphism is associated with a gain of function in P2X7 receptor and S. Hu1, F. Yu1, C. Ye1, X. Huang2, X. Lei1, Y. Dai3, H. Xu4, Y. Wang5, Y. Yu1

1Division of Rheumatology, 2Division of Gastroenterology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 3Division of Rheumatology, Wuhan PuAi Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 4Division of Endocrinology and Rheumatology, HuangPi People’s Hospital, 5State Key Laboratory of Natural Medicines, Department of Physiology, China Pharmaceutical University, Nanjing, China. Abstract receptor activity in the susceptibility of SLE with pericarditis in Chinese Han population.

Methods We studied a cohort of SLE patients with (SLE+PCS) or without (SLE-PCS) history of pericarditis and demographic, macrophages from whole blood by centrifugation on Ficoll gradient, in vitro macrophage releases of by LPS were evaluated by cytometric bead array. NLRP3 expression were evaluated by western blot after normalisation of . regression analysis was performed to evaluate the association of macrophages uptake of EB and NLRP3 expression.

Results demographic, disease activity and serological features were found between the two subgroups. The HWE analysis detected carrying wild type (CC). .

Conclusion .

Key words

Clinical and Experimental Rheumatology 2020; 38: 442-449. Clinical and Experimental Rheumatology 2020 P2RX7 SNP associated with SLE pericarditis / S. Hu et al.

Shaoxian Hu, MD Introduction Fei Yu, MD Pericarditis is one of the typical SLE SNPs could induce a gain of function Cong Ye, MD, PhD manifestations often associated with - Yujie Dai, MD - - chicine, whose action may be mediated by effect on microtubules during P2X7 Yikai Yu, MD, PhD This work should be attributed to been reported to be effective in treat- the Division of Rheumatology, ment for pericarditis in SLE and recur- The more relevant SNPs of Tongji Hospital, Tongji Medical College, Huazhong University of Science and and to decrease the rate of recurrence - Technology. - Please address correspondence The human P2X7 receptor is an ATP- ligand-gated cationic channel which the carboxyl terminal tail and leads to Yikai Yu Division of Rheumatology, has been shown to mediate the ATP-indifferent receptor function, as assessed Tongji Hospital, duced apoptotic death of dendritic cells, by ATP-induced Ca2+ monocytes and is mainly expressed on - District of Qiaokou, macrophages. The receptor is encoded perfunctional P2X7 receptor variants by a gene within human chromosome of ATP-gated transmembrane cation consequences of some SNPs channel P2X7 receptor in autoimmun- © Copyright CLINICAL AND ity has received particular attention due - EXPERIMENTAL RHEUMATOLOGY binant cells in vitro and in a Chinese In contrast to other P2X receptors, the P2X7 receptor C-terminal intracellular in particular carriers of A allele and AA/ chain is about 240 amino acids longer and is essential for its function. P2X7 - receptor is activated by high concen- trations of and prolonged exposure to - ATP. Once activated, it could undergo vious published studies and had shown the formation of a much larger pore a possible association between SNPs than any other P2X channel that allows and pericarditis in SLE patients carry- the passage of large cations, including SLE+PCS patients the presence of the functional consequences of this pore have been established for the P2X7 prompted expression of NLRP3, sug- receptors in the immune system. Pore gesting a possible involvement of P2X7 formation is necessary to trigger the in- receptor in the pathogenesis of SLE nate immune response induced by ATP, with pericarditis. Therefore, we carried a danger-associated molecular pattern out the case-control association study to - of the gene could be involved some activation and consequently cas- in the pathogenesis of activation of in- - carditis. Materials and methods which is associated with human SLE, Study participants so the functional polymorphisms of This is a cross-sectional study that in- this gene may affect SLE susceptibility. Several SNPs in the gene with history of pericarditis and 72 pa- a Wuhan-Chengguang grant. that affect the function of this recep- tients without pericarditis as a control ared. tor have been described. For instance, group. This cohort was set up from

443 P2RX7 SNP associated with SLE pericarditis / S. Hu et al.

individuals included in this cohort have Sequencing analysis. been followed up ever since. The diag- - es from a healthy donor were treated an independent ultrasound specialist. subject was determined in genomic control and washed twice with extracel- DNA samples. A SureSelect Human lular solution. - tion criteria for SLE. Disease activity solution enrichment of the coding ex- of macrophages determined scale by an independent nurse. SLE the manufacturer’s protocol. Adapter by western blot patients undergoing or had a biologic sequences for the Illumina HiSeq 2000 were ligated, and the enriched DNA not excluded in the present study. All samples were subjected to standard participants’ serum T-SPOT test were sample preparation. An Illumina CASA- were primed in the presence of 100 ng/ lysed in SDS sample buffer. Finally, Aligner algorithm was used to align cell-free supernatants were collected None of the patients had a history of sequence reads to the human reference genome and variants were analysed - - tein samples were loaded onto a 12.5% ing was performed by an independent genotype sequencing and divided into technician in a laboratory of medicine - at TongJi Hospital. - - cubated 2h with a commercial rabbit tee approved the study and written in- Macrophages release of IL-1 anti-human NLPR3 primary polyclonal formed consent was obtained from each patient. bead array were stripped and reprobed with a Isolation of peripheral blood mononuclear cells (PBMC) and were seeded in 24-well tissue culture - macrophages to adhere to the plastic plate for 2h at Finally, membranes were incubated of venous blood from each subject by a with a goat anti-rabbit secondary an- removed, and plates washed 2 times layered over 10ml of Ficoll-Histopaque peroxidase for 30 min. Labelled bands Then, the culture medium was replaced were visualised by the enhanced chemi- 2x10 supernatants were collected and stored glutamine, and 50U/ml penicillin and used for normalisation. order to enrich for macrophages, cells Uptake of ethidium bromide in freshly prepared macrophages Calculation of SLEDAI-2k score and 5% CO2, then washed two times 37°C for 5 min, in a solution contain- downloaded from Apple Store then registered, and an independent nurse

Genomic DNA isolation 2 completed the questionnaire with each - DAI in order to include persistent, ac- according to the manufacturer’s pro- the same conditions for an additional tive disease in those descriptors that tocol. DNA quality was determined by period of 10 min. Total plated cells had previously only considered new were counted with phase contrast, and permeabilised cells were counted with

444 Clinical and Experimental Rheumatology 2020 P2RX7 SNP associated with SLE pericarditis / S. Hu et al. disease in alopecia, mucous mem- Table I. Anthropometric and biochemical parameters of the SLE cohort population. brane ulcers, rash, and proteinuria to Variables SLE+PCS SLE-PCS p-value against the original SLEDAI as a pre- dictor for mortality and as a measure of global disease activity. It was also found to be equivalent to SLEDAI in describing changes in disease activity Statistical analysis 9 SNPs analysis of the genotype and al- 12 lele frequencies with comparisons be- 9 tween the two groups was performed using chi-square test by SPSS 10.1 Organ involvement software. Parametric analysis was performed using one-way analysis of vari- 0.97 the Hardy-Weinberg Equilibrium test 0.14 Current medication Wallis analysis was used when data were not normally distributed. The 0.43 Spearman linear correlation test was 0.54 performed to test the relative value of and macrophages permeability. The Hydroxychloroquin p-value was calculated based on two- 2=2.54, the time of the study. p

Results Genotype distribution in SLE cohort Demographic and clinical release from primed macrophages data analysis three possible genotypes SNP among in SLE cohort A total of 140 participants, among SLE cohort: the wild-type homozygous The hyperfunction of the P2X7 recep- CC as well as the heterozygous CT and tors induces the release of the natu- of pericarditis and 72 patients without homozygous TT, which presented the history of pericarditis enrolled in the study. Demographic and disease-relat- ed characteristics of the participants are as well as in SLE-PCS patients. Specif- explored the possible effect of the shown in Table I. One-way ANOVA for polymorphism of the gene on is 51.47% in SLE+PCS patients, while as arithmetic mean ± standard devia- - 2=5.02, p - between patients and controls with re- We then performed the Hardy-Wein- of auto-antibodies and complement 3 or 4.The biomedical parameters of sub- - jects have been presented in Table I. - - rophages among SLE cohort. Interest- of prednisone, or a combination with - or without immunomodulatory ther- tion. Homozygous genotype frequency in SLE-PCS patients.

Clinical and Experimental Rheumatology 2020 445 P2RX7 SNP associated with SLE pericarditis / S. Hu et al.

Table II. Assessment of frequency distribution of genotype polymorphism in the SLE cohort population. - rophages is higher than the others. Cor- p-value 2 relation analysis was performed and found that permeability of macrophag- 2 p2p - showed that in SLE+PCS cohort after challenging with ATP macrophages permeabilisation is positively associ- so we have made the hypothesis that after the P2X7 receptor activation, the

Discussion Fig. 1. The human P2X7 receptor is a double bearing variant functional polymorphism of transmembrane receptor which has a ubiquitous distribution and is most ex- post hoc test, n=3 experiments each in triplicate. *p clusively expressed on immune cells. repre- In macrophages and similar myeloid cells primed by lipopolysaccharide Expression of NLRP3 on ceptor. As we expected, after priming ef- extracellular ATP opens a cation chan- macrophages of SLE cohort by fect of LPS treatment on macrophages, +- as a consequence of increased NLRP3 sociated with processing and secretion In this part, we continued to evaluate if - - phism in SLE+PCS patients has effect - brain injury, data was shown that P2X7 - correlation of with NLRP3 expression - A the His155 to Thr SNP or a truncated - P2X7 splice variant that increases the activity showed no association with activity of this receptor have been re- p ported. It may produce allosteric chang- expression of each patient. Results are es in homomeric formations of P2X7 a representative of at least three inde- receptor subunits and pore protein- pendent experiments. NLRP3 expres- and regression study of macrophages folding structure. It has been reported sion was denoted by relative grey value permeabilisation in SLE+PCS patients - gain of function ofP2X7receptor, which lated macrophages. The results permea- affects macrophages from RA patients Correlation of NLRP3 with macrophages expressing P2X7 recep- SLEDAI-2K in SLE+PCS patients - After whole sequencing three possible - activated or highly expressed on mac- eters that may contribute permeability cohort: the wild-type homozygous CC rophages cells in autoimmune diseases. of macrophages in SLE+PCS patients, as well as the heterozygous CT and ho- - using freshly obtained or overnight cultured macrophages in SLE+PCS extracellular ATP acting at the P2X7 re- Hardy-Weinberg Equilibrium test and

Clinical and Experimental Rheumatology 2020 P2RX7 SNP associated with SLE pericarditis / S. Hu et al.

Fig. 2. A: Upper Lane: Wb detection of different samples lanes: Wb detection of house- B: • represents SLE patients and represents SLE patients without pericarditis

Fig. 3.AB p

receptor as a susceptibility factor in the contrary, in SLE-PCS patients no pathogenesis of pericarditis complicat- - ed with SLE has not been studied previ- masome expression and downstream SLE-PCS patients. As expected, a Chi- ously. The reported prevalence of pericarditis detected by echocardiographic studies, ranges from 11% to 54%, this present in SLE patients as well as in variability is partially attributable to the the possible effect of the healthy controls. We then performed the methods applied to document pericardi- - Chi-square test to compare the differ- al disease and whether symptomatic or some expression. Although the activity asymptomatic cases are included. In our of P2X7 receptor was regarded as in- SLE cohort, the prevalence of pericar- volved in the pathogenesis of autoim- ditis is 37.3% and our results show that mune diseases, including SLE and RA PCS subjects did. Thereafter, we detect- this receptor has been detected in different studies of SLE and the presence between pericarditis in SLE and SNP in SLE-PCS patients. In addition, the of SNP may account for the lost or gain p of function of the receptor. In our study, Then, we explored the possible effect observed in SLE+PCS individuals was we found that the expression of NLRP3 expression is strongly associated SNP gene on the levels of NLRP3 in- - released from macrophages and further tiprotein complex, which was formed - investigated the cellular permeability in - plication of pericarditis. SLE+PCS patients. These results suggest that underlying - - mechanisms might exist to modulate the activity of P2X7 receptor. In this re- pivotal mediators of the host response found increased expression of NLRP3 gard, we consider that it would be inter- to infections, tissue injury and immuno- on macrophages of SLE+PCS patients esting to assess the intracellular signal logic challenges. The role of the P2X7 pathways induced through P2X7 recep-

Clinical and Experimental Rheumatology 2020 447 P2RX7 SNP associated with SLE pericarditis / S. Hu et al.

macrophages compared with the wild would be promising to explore other mechanisms that could regulate function of P2X7 receptor, including its internalisation or the presence of micro RNAs that regulate expression of this receptor to further study the pathogenesis of the pericarditis in SLE.

Acknowledgments We are grateful to Dr Lu Yanjun from the central medical lab for technical as- sistance in genotyping and analysis.

References 1. E et al lupus erythematosus. Clin Exp Rheumatol 2. - : Successful treatment of re- current pleural and pericardial effusions with tocilizumab in a patient with systemic lupus erythematous 3. et al.: Inter- - carditis- ther Fig. 4. 4. et al.: shown in A and Colchicine: a simple and effective treatment Spearman correlation analysis was performed and p for pericarditis in systemic lupus erythema- A-D tosus? A report of 10 cases. Lupus 2015; 24: tor in immune cells from SLE patients, 5. et al.: Does mainly because different abnormalities colchicine decrease the rate of recurrence of acute idiopathic pericarditis treated with glu- have been described in other receptors - cocorticoids? of these cells. In our study it has been - response to ATP from macrophages of liaison. Front Pharmacol 7. : Purinergic SLE+PCS patients. When SLE subjects - signalling in autoimmunity: A role for the P2X7R in systemic lupus erythematosus? release. Conversely, in Silva’s research Biomed a higher incidence of complication of colchicine-pretreated macrophages cul- et al.: A novel mechanism of autophagic cell pericarditis, with an increased induc- tures could be observed approximately death in dystrophic muscle regulated by tion of pore formation was observed - P2RX7 receptor large-pore formation and HSP90. Autophagy 2015; 11: 113-30. 9. PÉTRILLI V, PAPIN S, DOSTERT C et al.: Acti- - In the study we have made a prelimi- gered by low intracellular potassium concen- adhesion and migration of immune - tration. Cell Death Differ 10. et al.: A His-155 to Tyr polymorphism confers gain-of-function to the human P2X7 receptor expression analysis as the negative activation in SLE+PCS patients. Por- tale’s and our study showed that the of colchicine is notable and it underlies 11. et al.: to susceptibility to SLE indifferent eth- polymorphism of P2RX7 gene confers an enhanced function of P2X7 receptor in immune - cells from patients with rheumatoid arthritis. - genotype Cell Immunol 12. et al.: bule rearrangement induced by ATP via - P2X7 receptors, consequently reducing

Clinical and Experimental Rheumatology 2020 P2RX7 SNP associated with SLE pericarditis / S. Hu et al.

exhibit a gain-of-function effect and en- . 21. : Colchicine in pericardial disease: from the underlying biology 17. : - 13. Summarizing disease features over time: II. tions in cardiovascular medicine. Curr Car- et al.: A Thr357 to Ser polymorphism in ho- - diol Rep mozygous and compound heterozygous sub- matol 22. et al.: jects causes absent or reduced P2X7 function et - - al- ing by macrophages complicated with amyloidosis. Mod Rheu- . matol 14. et al.: Dis Model Mech 23. et al.: 19. et al.: P2X7R - of function of the human P2X7 receptor. duced by ATP in P2X2 and P2X7 receptor-ex- activation and brain injury in a rat model of pressing cells: implications for its therapeutic 15. et al.: Association of intracerebral hemorrhage: involvement of action P2X7R gene polymorphisms with systemic peroxynitrite 2015; 12: 24. et al.: Expres- lupus erythematosus in a Chinese population. 190-207. Mutagenesis 20. : Colchicine: its mechanism of action receptor in patients with systemic lupus ery- et al.: - thematosus and rheumatoid arthritis. Hum - tion. Curr Rheumatol Rep Immunol

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