P2X7-Mediated Antigen-Independent Activation of CD8+ T Cells Promotes Salt-Sensitive Hypertension

Abstract

Background: CD8⁺ T cells (CD8Ts) have been implicated in hypertension. However, the specific mechanisms are not fully understood. In this study, we explore the contribution of the P2X7 (purinergic receptor P2X7) receptor to CD8T activation and subsequent promotion of sodium retention in the kidney.

Methods: We used mouse models of hypertension. Wild type were used as genetic controls, OT1 and Rag2/OT1 mice were utilized to determine antigen dependency, and P2X7-knockout mice were studied to define the role of P2X7 in activating CD8Ts and promoting hypertension. Blood pressure was monitored continuously and kidneys were obtained at different experimental end points. Freshly isolated CD8Ts from mice for activation assays and ATP stimulation. CD8T activation-induced promotion of sodium retention was explored in cocultures of CD8Ts and mouse DCTs.

Results: We found that OT1 and Rag2/OT1 mice, which are nonresponsive to common antigens, still developed hypertension and CD8T-activation in response to deoxycorticosterone acetate/salt treatment, similar to wild-type mice. Further studies identified the P2X7 receptor on CD8Ts as a possible mediator of this antigen-independent activation of CD8Ts in hypertension. Knockout of the P2X7 receptor prevented calcium influx and cytokine production in CD8Ts. This finding was associated with reduced CD8T-DCT stimulation, reversal of excessive salt retention in DCTs, and attenuated development of salt-sensitive hypertension.

Conclusions: Our findings suggest a novel mechanism by which CD8Ts are activated in hypertension to exacerbate salt retention and infer that the P2X7 receptor on CD8Ts may represent a new therapeutic target to attenuate T-cell-mediated immunopathology in hypertension.

Keywords: blood pressure; cytokines; hypertension; kidney; sodium chloride.

Figure 1:. Rag2/OT1 mice exhibit hypertension, T-cell activation and renal infiltration upon DOCA/salt treatment, despite the lack of antigen.

A) Radio-biotelemetry was used to continuously monitor BP in Rag2/OT1 mice treated with DOCA/salt. Average systolic BP of baseline (days 0–3, Base, Blue) and endpoint (days 16–19, End, Red) were compared using t-test, n=5–6 per group. B) Flow cytometry study to examine T cells infiltrated into the kidney of Sham or DOCA/salt-treated Rag2/OT1 mice at the end point of a. n=5–6, gating strategy include singlets – cells – FVD – CD3, as shown in Figure S3. C) Flow cytometry was used to identify CD44-positive OT1 T cells in the spleens of Sham or DOCA-treated Rag2/OT1 mice. quantification compared using t-test, n=5–6, gating strategy shown in Figure S3. D) Cytokine production was determine in OT1 T cells isolated from hypertensive or normotensive Rag2/OT-1 mice using realtime-PCR. Data analyzed by t-test, n = 5–6 per group.

Figure 2:. Extracellular ATP stimulates cytokine production in WT but not P2X7 KO CD8Ts.

A) Mouse CD8Ts were stimulated by 40 mM NaCl, 10 μM Norepinephrine, 100 nM angiotensin II, 100 nM aldosterone, 1 mM ATP, or 100 μM BzATP for 3 hours and IFNγ transcription was measured using rt-qPCR, data normalized to β-actin. Changes in IFNγ transcription were quantified by Brown-Forsythe ANOVA (P = 0.0018) followed by post hoc Dunnett’s T3 comparing each treatment group to the control. B-E) Primary isolated splenic CD8Ts from WT (B, D) or P2X7 KO (C, E) mice were treated for 3 hours with ATP or BzATP and analyzed via rt-qPCR for IFNγ and TNFα transcription, normalized to β-actin. *P < 0.05, **P < 0.01, ***P <0.001, ****P < 0.0001

Figure 3:. Extracellular ATP treatment increases calcium influx in WT CD8Ts but not P2X7 KO CD8Ts.

Splenic CD8Ts from WT or P2X7 KO mice were pre-loaded with Fluo-4 dye and then stimulated with the P2X7 agonist BzATP (A, B) or ATP (C, D). Calcium influx in cells was analyzed by flow cytometry and quantitated by geometric mean fluorescent intensity (gMFI). Panel colors are indicative of recorded population density. Fluorescence from cells in HBSS (n= 5–7 per group) or calcium-free media (n = 3–4 per group) were quantified in 200-second segments in B) and D). Data were analyzed by Two-way ANOVA via mixed effect analysis followed by posthoc Sidak’s for comparison of gMFI at each time point for HBSS groups, and Dunnett’s for comparison of gMFI at each time point with baseline in calcium-free treatment groups. ns = not significant. *P < 0.05, **P < 0.01, ***P <0.001. Data are expressed ± SEM, with no visible error bars in d) due to small variability.

Figure 4:. Pre-activation of CD8Ts with the P2X7 agonist results in increased NCC-mediated sodium retention in co-cultured DCTs.

A) Schematic of the co-culture experimental protocol. B) Gating strategy for mDCTs after selection and staining. C) Representative flow cytometry images showing an increase in CoroNa Green (measuring intracellular Na⁺) and PDL1 in mDCTs following co-culture with pre-stimulated CD8Ts compared to naïve CD8Ts. Quantified in D-F) with gating as shown for gMFI calculation (grey box, D, E) or percentage of both high intracellular sodium and high PDL1 (F). Analyzed via unpaired t-tests.

Figure 5:. Expression of P2X7 in CD8Ts and the impact of P2X7 knockout in DOCA/salt-induced salt-sensitive hypertension.

A) Representative Simple Western for P2X7 expression in CD8Ts from WT mice treated with DOCA/salt or sham, P = 0.0324. B) Radiotelemetry recording of systolic BP in WT (grey trace) and P2X7 KO (blue trace) mice receiving DOCA/salt treatment. Seven days after DOCA/salt initiation, P2X7 KO mice were randomly selected to receive saline (remain blue trace) or naïve WT CD8Ts (red trace). C) Quantification of BP in b) at days 0–1 (baseline), 9–10 (7 days post-DOCA/salt, before restoring WT CD8Ts), and 17–18 (7 days after adoptive transfer of WT CD8Ts). D) Quantifying T cells in kidneys taken from experimental P2X7 KO mice at the study endpoint. Each dot represents the mean of 4 images taken per kidney. See Figure S10 for representative images. For BP and T cell counting data, data analyzed via t-test or one-way ANOVA followed by posthoc Tukey’s. ns = not significant, * P < 0.05, ** P < 0.01, *** P <0.001. E) Kidneys obtained from P2X7 KO DOCA or P2X7 KO DOCA + WT naïve CD8T mice at the experimental endpoint (see Figure 5b) were stained and quantified for NCC expression. Each dot represents the mean intensity values of 4 images taken per kidney section, P = 0.00002502.

Figure 6:. P2X7 deficient CD8Ts are not activated in DOCA/salt model and failed to cause salt-sensitive hypertension.

A) CD8Ts were isolated from the spleens of P2X7 KO mice and treated for 3 hours with PMA & Ionomycin in the presence of Brefeldin A, stained for intracellular IFNγ and TNFα production and analyzed via flow cytometry. Quantified in B). C) Representative flow cytometry images of mDCTs co-cultured with P2X7 KO CD8Ts isolated from DOCA/salt- or sham-treated mice and analyzed for intracellular sodium using CoroNa Green. D) Systolic BP in P2X7 KO mice that received adoptive transfer of CD8Ts isolated from spleens of DOCA/salt-treated WT mice. E) Systolic BP in WT mice that received adoptive transfer of CD8Ts isolated from spleens of DOCA/salt-treated P2X7 KO mice. Mean three-day baseline systolic BP was compared with the last three-day mean systolic BP via an unpaired t-test. ns = not significant, **P < 0.01, ***P < 0.001, r = recipient, d = donor