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ATP release drives heightened immune responses associated with hypertension

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Science Immunology  28 Jun 2019:
Vol. 4, Issue 36, eaau6426
DOI: 10.1126/sciimmunol.aau6426
  • Fig. 1 Antigen-specific immune responses are enhanced in hypertension.

    (A and B) Normotensive and Ang II–induced hypertensive C57BL/6 mice were immunized subcutaneously (s.c.) with OVA-CFA (A) or intraperitoneally (i.p.) with OVA-LPS (B). Seven days later, the percentages and numbers of OVA-specific T cells among total CD8+ T cells in blood (A) and spleen (B) were determined by SIINFEKL–H-2Kb tetramer staining. (C) Mice were immunized intraperitoneally with OVA-alum. Seven days later, their splenocytes were restimulated with SIINFEKL peptide. IL-2 and IFN-γ secretion were measured after 24 and 72 hours, respectively, by ELISA. (D) Seven days after the immunization with OVA-CFA or OVA-alum, splenocytes were restimulated with the MHC class II dominant peptide OVA323–339. Secretion of IL-2 was measured at 24 hours by ELISA. Data are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005.

  • Fig. 2 Hypertensive mice are predisposed to autoimmune diseases.

    (A to C) RIP-mOVA mice were made hypertensive by treatment with either Ang II or L-NAME. After 2 to 3 weeks, when hypertension was established, 5 × 106 OT-I T cells (CD8+ T cells from OT-I transgenic mice) were transferred intravenously into normotensive and hypertensive RIP-mOVA mice. (A) Blood glucose levels were measured for 2 weeks after OT-I T cell transfer. (B and C) Two weeks after OT-I T cell transfer, the percentages of OT-I T cells among CD8+ T cells in the blood, spleen, and pancreatic draining lymph nodes (DLN) were quantified by tetramer analysis (B), and the numbers of inflamed islets, identified by anti-CD3 staining, were counted in a blinded fashion (C). (D to F) ConA (5 mg/kg) was injected intravenously into normotensive and hypertensive mice. Blood ALT levels were measured after 6 hours. (D) Hepatic inflammatory cells were prepared by tissue enzymatic digestion followed by Percoll centrifugation. (E) Cells were cultured in medium for 6 hours in the presence of brefeldin A, and then intracellular staining was performed to examine the production of IFN-γ and TNF-α by CD4+ T cells. Liver necrosis area was measured after hematoxylin and eosin staining. MFI, mean fluorescence intensity. (F) For each liver, necrosis area represents the average of 10 separate fields. In (A) to (C), n = 17, 6, and 12 for normotensive, Ang II–treated, and L-NAME–treated mice, respectively. Data are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005.

  • Fig. 3 APCs from hypertensive mice present antigens more efficiently.

    (A and B) Splenic DCs and peritoneal macrophages (PM) from normotensive or Ang II–induced hypertensive C57BL/6 mice were pulsed with 10 μM OVA (A) or 50 pM SIINFEKL peptide (SKL) (B) in vitro and were then coincubated with OT-I T cells. The percentages of CD69+ (after 4 hours) or IFN-γ+ cells (after 6 hours in the presence of brefeldin A) among OT-I T cells were measured by flow cytometry. Representative flow cytometry dot plots of IFN-γ and CD69 expression are shown. (C) Splenic DCs from normotensive or L-NAME–induced hypertensive mice were pulsed with SIINFEKL and then coincubated with OT-I T cells. The percentages of CD69+ or IFN-γ+ cells among all CD8+ T cells were measured. (D) Splenic DCs from normotensive or Ang II–induced hypertensive mice were loaded with SIINFEKL for 4 hours ex vivo. Cells were then transferred intravenously into naïve C57BL/6 mice. Seven days later, splenocytes from the recipients were restimulated with SIINFEKL and the secretion of IL-2 and IFN-γ was measured. Data are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005.

  • Fig. 4 CD86 is up-regulated on the APCs of hypertensive mice.

    C57BL/6 mice were sham-treated (NT) or Ang II–treated (HT) for 2 weeks. (A) Surface CD80 and CD86 expression on splenic DCs and peritoneal macrophages (Mφ). Surface expression of CD86 by Kupffer cells was also measured. The representative histograms are shown. (B) DCs and macrophages from naïve mice were stimulated in vitro with or without 10 μM Ang II overnight, and surface CD86 expression was measured. (C) Mice were either sham-treated or treated with Ang II and hydralazine (Hyd) for 14 days. Surface CD86 expression was measured in splenic DCs and peritoneal macrophages. Data are means ± SEM. **P < 0.01; ***P < 0.005.

  • Fig. 5 Blocking CD86 eliminates the overactivation of immune responses in hypertensive mice.

    C57BL/6 mice were sham-treated (normotensive) or Ang II–treated (hypertensive) for 2 weeks. (A) Splenic DCs were purified. Some of the cells were pulsed with SIINFEKL peptide. Some cells were also treated with an anti-CD80 antibody or an anti-CD86 antibody or both. They were then coincubated with OT-I T cells, and the expression of T cell CD69 and IFN-γ was measured. (B and C) Mice were regularly treated with an anti-CD86 antibody or an isotype control antibody from 3 days before being immunized with OVA until sacrifice. Seven days after immunization, the numbers of OVA-specific CD8+ T cells in the blood (B) and splenocyte IL-2 production after SIINFEKL restimulation were measured (C). (D and E) Acute hepatitis was induced by intravenous injection of ConA (5 mg/kg) into normotensive and hypertensive mice. Some hypertensive mice were injected intraperitoneally with either CD86 blocking antibody (α-CD86) or an isotype control antibody (IgG) every other day for three times before ConA administration. (D) Blood ALT levels were measured 6 hours after ConA injection. (E) Leukocytes in the liver were isolated with Percoll gradients. Cells were cultured in medium for 6 hours in the presence of brefeldin A, and then intracellular staining was performed to examine the production of IFN-γ and TNF-α by CD4+ T cells. Data are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005. NS, not significant.

  • Fig. 6 ATP is elevated early in hypertension and is responsible for CD86 up-regulation through the P2X7 receptor in APCs.

    (A) DAMPs including ATP, uric acid, oxLDL, and HMGB-1 were measured in the plasma of normotensive mice, and mice were made hypertensive for 6 days. (B) Blood plasma ATP levels were measured at different time points after hypertension induction. A group of mice were cotreated with Ang II and hydralazine [14d(Hyd)]. n = 5 for normotensive, Ang II–treated, and Ang II and hydralazine cotreated mice; n = 4 for L-NAME–treated mice. (C) Surface CD86 expression on splenic DCs from mice infused with Ang II for 0, 4, 7, or 14 days. (D) Splenic DCs from naïve mice were stimulated with the indicated concentrations of ATP for 12 hours in vitro, and their surface CD86 expression was measured. (E) Splenic DCs were stimulated with medium or 3 μM ATP. Some ATP-treated cells were also incubated with antagonists to P2X7 [A740003 (A74) or BBG], pan-P2Y (suramin), P2Y2 [AR-C 118925XX (AR-C)], or P2Y1 (BPTU). Surface CD86 was measured after 18 hours. (F) Splenic DCs from P2X7-deficient mice were treated with different concentrations of ATP, and surface CD86 was measured. (G) Surface CD86 expression on splenic DCs and peritoneal macrophages was derived from normotensive and hypertensive P2X7-deficient mice.

  • Fig. 7 Activation of the P2X7 receptor on APCs by 3 μM ATP initiates the heightened immune responses associated with hypertension.

    (A) Splenocytes from either C57BL/6 or P2X7 knockout mice were labeled with the Ca2+ probe Fluo-4 AM in vitro. The cells were then treated with either 3 or 500 μM ATP, and Ca2+ influx into DCs (left) and T cells (middle) was monitored continuously for 4 min by flow cytometry (right). The first minute kinetics of Ca2+ influx into DCs in the presence of 3 μM ATP is also shown. (B) Splenic DCs were stimulated with or without 3 μM ATP. Some of the ATP-treated cells were pretreated with the calcium chelator BAPTA or the calmodulin inhibitor chlorpromazine (Chlor). Surface CD86 was measured after 18 hours. (C) Peritoneal macrophages were treated with 3 or 500 μM ATP for 18 hours ex vivo, and IL-1β in the medium was measured by ELISA. Data were analyzed with paired Student’s t test. (D) Splenic DCs and T cells were purified from the spleen of naïve mice. DCs (2 × 106), T cells, and HEK-293 cells were pelleted and then dissolved in 100 μl of acetonitrile. The concentrations of LPCs C16:0 and C18:0 were determined by LC-MS/MS. (E) Concentrations of LPCs C16:0 and C18:0 in the plasma of normotensive and hypertensive mice. (F) Splenic DCs from normotensive and hypertensive mice were treated with vehicle, apyrase, or A740003 for 18 hours. Surface expression of CD86 was then measured. (G) Normotensive and hypertensive mice were pretreated intraperitoneally with apyrase (0.2 U/g weight) or A740003 (50 nmol/mouse) every other day for 6 days, and they were then immunized with OVA-CFA. Apyrase or A740003 was continued after immunization. The percentages of OVA-specific CD8+ T cells in the blood were evaluated 7 days after immunization. (H and I) Normotensive and hypertensive mice were treated with ConA to induce hepatitis with or without the P2X7 receptor antagonist A740003. Plasma ALT levels (H) and CD4+ T cell cytokines (I) were measured.

  • Fig. 8 The correlation of plasma ATP levels and P2X7 receptor expression with BP in mice and humans.

    (A) BP of P2X7 knockout mice was made hypertensive with Ang II (left) or L-NAME (right). Two-way ANOVA with Bonferroni’s correction was used for BP data analysis. (B) Aorta, renal arteries, and total blood cells were collected from normotensive mice and hypertensive mice treated with Ang II for 2 weeks. Some blood cells were further separated to isolate leukocytes and erythrocytes with Percoll gradients. The arteries and cells were placed in serum-free medium for 3 hours, and the ATP levels in the medium were measured. (C) Distribution of plasma ATP in hypertensive patients and normotensive controls. The Kruskal-Wallis nonparametric test was used for analysis. (D) Distribution of plasma ATP in normotensive people (NTN), hypertensive patients with BPs not well controlled (HTN), and hypertensive patients with BP controlled to normal levels (C-HTN). Wilcoxon rank sum test (LSD test adjusted) was used for analysis. (E) Fit plots between ATP and SBP (left) and between ATP and DBP (right) among all subjects. *P < 0.05; **P < 0.01; ***P < 0.005.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/36/eaau6426/DC1

    Fig. S1. BP measurements after hypertension induction in mice.

    Fig. S2. Schematic diagram for RIP-mOVA mice diabetes study.

    Fig. S3. ConA-induced hepatitis model.

    Fig. S4. TCR sensitivity is not altered after hypertension.

    Fig. S5. The phenotypes of hypertensive DCs.

    Fig. S6. Proinflammatory cytokines produced by hypertensive APCs.

    Fig. S7. Expression of CD86 and CD80.

    Fig. S8. Calcium influx under ATP stimulation.

    Fig. S9. ROS and PGE2 production by DCs after 3 μM ATP treatment.

    Fig. S10. ATP-induced impermeable dye (YO-PRO-1) uptake and apoptosis.

    Fig. S11. ATP inhibition and APC surface CD86.

    Fig. S12. P2X7-deficient DC transfer.

    Fig. S13. Hypertension induction in the presence of P2X7 antagonist.

    Fig. S14. Gating strategies of flow cytometry analyses.

    Table S1. The characteristics of hypertensive patients and normotensive controls.

    Table S2. The characteristics of normotensive people (NTN), hypertensive patients with BP not well controlled (HTN), and hypertensive patients with BP controlled within normal levels (C-HTN).

    Table S3. Multivariate linear regressions between plasma ATP and SBP.

    Table S4. Multivariate linear regressions between plasma ATP and DBP.

    Table S5. Raw data file.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. BP measurements after hypertension induction in mice.
    • Fig. S2. Schematic diagram for RIP-mOVA mice diabetes study.
    • Fig. S3. ConA-induced hepatitis model.
    • Fig. S4. TCR sensitivity is not altered after hypertension.
    • Fig. S5. The phenotypes of hypertensive DCs.
    • Fig. S6. Proinflammatory cytokines produced by hypertensive APCs.
    • Fig. S7. Expression of CD86 and CD80.
    • Fig. S8. Calcium influx under ATP stimulation.
    • Fig. S9. ROS and PGE2 production by DCs after 3 μM ATP treatment.
    • Fig. S10. ATP-induced impermeable dye (YO-PRO-1) uptake and apoptosis.
    • Fig. S11. ATP inhibition and APC surface CD86.
    • Fig. S12. P2X7-deficient DC transfer.
    • Fig. S13. Hypertension induction in the presence of P2X7 antagonist.
    • Fig. S14. Gating strategies of flow cytometry analyses.
    • Table S1. The characteristics of hypertensive patients and normotensive controls.
    • Table S2. The characteristics of normotensive people (NTN), hypertensive patients with BP not well controlled (HTN), and hypertensive patients with BP controlled within normal levels (C-HTN).
    • Table S3. Multivariate linear regressions between plasma ATP and SBP.
    • Table S4. Multivariate linear regressions between plasma ATP and DBP.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S5 (Microsoft Excel format). Raw data file.

    Files in this Data Supplement:

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