Angiotensin II type-1 receptor (AT1R) regulates expansion, differentiation, and functional capacity of antigen-specific CD8+ T cells

Angiotensin II (Ang II) and its receptor AT1 (AT1R), an important effector axis of renin-angiotensin system (RAS), have been demonstrated to regulate T-cell responses. However, these studies characterized Ang II and AT1R effects using pharmacological tools, which do not target only Ang II/AT1R axis. The specific role of AT1R expressed by antigen-specific CD8+ T cells is unknown. Then we immunized transgenic mice expressing a T-cell receptor specific for SIINFEKL epitope (OT-I mice) with sporozoites of the rodent malaria parasite Plasmodium berghei expressing the cytotoxic epitope SIINFEKL. Early priming events after immunization were not affected but the expansion and contraction of AT1R-deficient (AT1R−/−) OT-I cells was decreased. Moreover, they seemed more activated, express higher levels of CTLA-4, PD-1, LAG-3, and have decreased functional capacity during the effector phase. Memory AT1R−/− OT-I cells exhibited higher IL-7Rα expression, activation, and exhaustion phenotypes but less cytotoxic capacity. Importantly, AT1R−/− OT-I cells show better control of blood parasitemia burden and ameliorate mice survival during lethal disease induced by blood-stage malaria. Our study reveals that AT1R in antigen-specific CD8+ T cells regulates expansion, differentiation, and function during effector and memory phases of the response against Plasmodium, which could apply to different infectious agents

Group V secretory phospholipase A2 is involved in tubular integrity and sodium handling in the kidney

Group V (GV) phospholipase A2 (PLA2) is a member of the family of secreted PLA2 (sPLA2) enzymes. This enzyme has been identified in several organs, including the kidney. However, the physiologic role of GV sPLA2 in the maintenance of renal function remains unclear. We used mice lacking the gene encoding GV sPLA2 (Pla2g5−/−) and wild-type breeding pairs in the experiments. Mice were individually housed in metabolic cages and 48-h urine was collected for biochemical assays. Kidney samples were evaluated for glomerular morphology, renal fibrosis, and expression/activity of the (Na+ + K+)-ATPase α1 subunit. We observed that plasma creatinine levels were increased in Pla2g5−/− mice following by a decrease in creatinine clearance. The levels of urinary protein were higher in Pla2g5−/− mice than in the control group. Markers of tubular integrity and function such as γ-glutamyl transpeptidase, lactate dehydrogenase, and sodium excretion fraction (FENa+) were also increased in Pla2g5−/− mice. The increased FENa+ observed in Pla2g5−/− mice was correlated to alterations in cortical (Na+ + K+) ATPase activity/ expression. In addition, the kidney from Pla2g5−/− mice showed accumulation of matrix in corticomedullary glomeruli and tubulointerstitial fibrosis. These data suggest GV sPLA2 is involved in the maintenance of tubular cell function and integrity, promoting sodium retention through increased cortical (Na+ + K+)-ATPase expression and activity

Impairment of the Plasmodium falciparum Erythrocytic Cycle Induced by Angiotensin Peptides

Plasmodium falciparum causes the most serious complications of malaria and is a public health problem worldwide with over 2 million deaths each year. The erythrocyte invasion mechanisms by Plasmodium sp. have been well described, however the physiological aspects involving host components in this process are still poorly understood. Here, we provide evidence for the role of renin-angiotensin system (RAS) components in reducing erythrocyte invasion by P. falciparum. Angiotensin II (Ang II) reduced erythrocyte invasion in an enriched schizont culture of P. falciparum in a dose-dependent manner. Using mass spectroscopy, we showed that Ang II was metabolized by erythrocytes to Ang IV and Ang-(1–7). Parasite infection decreased Ang-(1–7) and completely abolished Ang IV formation. Similar to Ang II, Ang-(1–7) decreased the level of infection in an A779 (specific antagonist of Ang-(1–7) receptor, MAS)-sensitive manner. 10−7 M PD123319, an AT2 receptor antagonist, partially reversed the effects of Ang-(1–7) and Ang II. However, 10−6 M losartan, an antagonist of the AT1 receptor, had no effect. Gs protein is a crucial player in the Plasmodium falciparum blood cycle and angiotensin peptides can modulate protein kinase A (PKA) activity; 10−8 M Ang II or 10−8 M Ang-(1–7) inhibited this activity in erythrocytes by 60% and this effect was reversed by 10−7 M A779. 10−6 M dibutyryl-cAMP increased the level of infection and 10−7 M PKA inhibitor decreased the level of infection by 30%. These results indicate that the effect of Ang-(1–7) on P. falciparum blood stage involves a MAS-mediated PKA inhibition. Our results indicate a crucial role for Ang II conversion into Ang-(1–7) in controlling the erythrocytic cycle of the malaria parasite, adding new functions to peptides initially described to be involved in the regulation of vascular tonus

Impairment of the Plasmodium falciparum Erythrocytic Cycle Induced by Angiotensin Peptides

Plasmodium falciparum causes the most serious complications of malaria and is a public health problem worldwide with over 2 million deaths each year. The erythrocyte invasion mechanisms by Plasmodium sp. have been well described, however the physiological aspects involving host components in this process are still poorly understood. Here, we provide evidence for the role of renin-angiotensin system (RAS) components in reducing erythrocyte invasion by P. falciparum. Angiotensin II (Ang II) reduced erythrocyte invasion in an enriched schizont culture of P. falciparum in a dose-dependent manner. Using mass spectroscopy, we showed that Ang II was metabolized by erythrocytes to Ang IV and Ang-(1–7). Parasite infection decreased Ang-(1–7) and completely abolished Ang IV formation. Similar to Ang II, Ang-(1–7) decreased the level of infection in an A779 (specific antagonist of Ang-(1–7) receptor, MAS)-sensitive manner. 10−7 M PD123319, an AT2 receptor antagonist, partially reversed the effects of Ang-(1–7) and Ang II. However, 10−6 M losartan, an antagonist of the AT1 receptor, had no effect. Gs protein is a crucial player in the Plasmodium falciparum blood cycle and angiotensin peptides can modulate protein kinase A (PKA) activity; 10−8 M Ang II or 10−8 M Ang-(1–7) inhibited this activity in erythrocytes by 60% and this effect was reversed by 10−7 M A779. 10−6 M dibutyryl-cAMP increased the level of infection and 10−7 M PKA inhibitor decreased the level of infection by 30%. These results indicate that the effect of Ang-(1–7) on P. falciparum blood stage involves a MAS-mediated PKA inhibition. Our results indicate a crucial role for Ang II conversion into Ang-(1–7) in controlling the erythrocytic cycle of the malaria parasite, adding new functions to peptides initially described to be involved in the regulation of vascular tonus

AT1 receptor-mediated angiotensin II activation and chemotaxis of T lymphocytes

Angiotensin II (Ang II), a central renin–angiotensin system (RAS) effector molecule, and its receptors, AT1 and AT2, have been shown to be involved in the inflammatory aspects of different diseases, however the cellular mechanisms underlying the regulation of immunity are not fully understood. In this work, using spleen-derived CD4+ and CD8+ T lymphocytes activated in vitro, we tested the influence of Ang II on different aspects of the T cell function, such as activation and adhesion/transmigration through endothelial basal membrane proteins. The addition of 10−8 M Ang II did not change any of the parameters evaluated. However, 10−6 M losartan, an AT1 receptor antagonist: (i) reduced the percentage of CD25+ and CD69+ cells of both subsets; (ii) inhibited adhesion of these cells to fibronectin or laminin by 53% or 76%, respectively and (iii) significantly reduced transmigration through fibronectin or laminin by 57% or 43%, respectively. In addition, 10−6 M captopril, an angiotensin-converting enzyme inhibitor had similar effects to Ang II, however its effects were reverted by exogenous Ang II (10−8 M). None of these responses was modified by 10−7 M PD123319, an AT2 antagonist. These data reinforce the notion of endogenous production of Ang II by T cells, which is important for T cell activation, and adhesion/transmigration induced on interaction with basal membrane proteins, possibly involving AT1 receptor activation. Moreover, AT1 receptor expression is 10-fold higher in activated T lymphocytes compared with naive cells, but AT2 receptor expression did not change after T cell receptor triggering

Regulation of extracellular ATP in human erythrocytes infected with Plasmodium falciparum.

In human erythrocytes (h-RBCs) various stimuli induce increases in [cAMP] that trigger ATP release. The resulting pattern of extracellular ATP accumulation (ATPe kinetics) depends on both ATP release and ATPe degradation by ectoATPase activity. In this study we evaluated ATPe kinetics from primary cultures of h-RBCs infected with P. falciparum at various stages of infection (ring, trophozoite and schizont stages). A "3V" mixture containing isoproterenol (β-adrenergic agonist), forskolin (adenylate kinase activator) and papaverine (phosphodiesterase inhibitor) was used to induce cAMP-dependent ATP release. ATPe kinetics of r-RBCs (ring-infected RBCs), t-RBCs (trophozoite-infected RBCs) and s-RBCs (schizont-infected RBCs) showed [ATPe] to peak acutely to a maximum value followed by a slower time dependent decrease. In all intraerythrocytic stages, values of ΔATP1 (difference between [ATPe] measured 1 min post-stimulus and basal [ATPe]) increased nonlinearly with parasitemia (from 2 to 12.5%). Under 3V exposure, t-RBCs at parasitemia 94% (t94-RBCs) showed 3.8-fold higher ΔATP1 values than in h-RBCs, indicative of upregulated ATP release. Pre-exposure to either 100 µM carbenoxolone, 100 nM mefloquine or 100 µM NPPB reduced ΔATP1 to 83-87% for h-RBCs and 63-74% for t94-RBCs. EctoATPase activity, assayed at both low nM concentrations (300-900 nM) and 500 µM exogenous ATPe concentrations increased approx. 400-fold in t94-RBCs, as compared to h-RBCs, while intracellular ATP concentrations of t94-RBCs were 65% that of h-RBCs. In t94-RBCs, production of nitric oxide (NO) was approx. 7-fold higher than in h-RBCs, and was partially inhibited by L-NAME pre-treatment. In media with L-NAME, ΔATP1 values were 2.7-times higher in h-RBCs and 4.2-times higher in t94-RBCs, than without L-NAME. Results suggest that P. falciparum infection of h-RBCs strongly activates ATP release via Pannexin 1 in these cells. Several processes partially counteracted ATPe accumulation: an upregulated ATPe degradation, an enhanced NO production, and a decreased intracellular ATP concentration

Effect of Pannexin 1 inhibitors on [ATPe] kinetics of a highly enriched population of trophozoite infected erythrocytes.

<p>A. The time course of [ATPe] (pmol/10⁶ cells) was assessed for trophozoite-infected erythrocytes at 94% parasitemia (denoted as t94-RBCs) in the absence and presence of 100 µM carbenoxolone (CBX), 100 nM mefloquine (MFQ), or 100 µM of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), inhibitors of Pannexin 1. Exposure to 3V is indicated by the arrow. In some experiments, prior to 3V exposure cells were pre-incubated 10 min with either CBX, MFQ or NPPB. For a comparison, similar experiments with noninfected RBCs (h-RBCs) are shown. t94-RBCs (N = 14, n = 19), t94-RBCs +CBX (N = 6, n = 7), t94-RBCs+MFQ (N = 4, n = 4), t94-RBCs+NPPB (N = 4, n = 4), h-RBCs (N = 15; n = 19), h-RBCs+CBX (N = 6, n = 9), h-RBCs+MFQ (N = 4, n = 4), h-RBCs+NPPB (N = 3, n = 3). N = independent preparations, n = replicates. B. 3V-dependent increase of [ATPe] calculated from A. Values are expressed as ΔATP₁, i.e., the difference between [ATPe] at 1 min post-stimulus and basal [ATPe]. Results are means ± SEM. (*p<0.05, ***p<0.001).</p

3V-dependent ATP kinetics of <i>P. falciparum</i> infected RBCs.

<p>A, C. Time course of ATPe concentration (pmol/10⁶ cells) for r-RBCs (ring-infected erythrocytes), t-RBCs (trophozoite-infected erythrocytes) and s-RBCs (schizont-infected erythrocytes) at low parasitemia (<5%, A) and high parasitemia (5–12.5%, C). In the time indicated by the arrow, cells were exposed to “3V”, a cAMP activating cocktail containing 10 mM isoproterenol, 30 mM forskolin and 100 mM papaverine. For a comparison, similar experiments with h-RBCs are shown. B, D. 3V-dependent increases of [ATPe] calculated from A and C. Values are expressed as ΔATP₁ for low parasitemia (B) and high parasitemia (D). Results are means ± SEM. (*p<0.05, ***p<0.001). (N, n), with N = independent preparations, n = replicates. E. Initial rate of [ATPe] decay (pmol/10⁶ cells/min) taken from data of C.</p

3V-dependent increase of [ATPe] of trophozoite-infected RBCs.

<p>Values of ΔATP₁ as a function of parasitemia (5–12.5%) for trophozoite-infected RBCs (N = 4, n = 4–5). Prior to experiments, cells were pre-incubated 3 hours in the absence (black circles) or presence (green squares) of 2 mM L-NAME. Hyperbolic functions were fitted to experimental data. Results are means ± SEM with N = 3 and n = 5–10. N = independent preparations, n = replicates. The red symbols illustrate an estimate of ΔATP₁ under a hypothetical situation where ectoATPase activity is blocked. It was calculated by: 1- estimating the concentration of ATPe hydrolyzed during the first minute post-stimulus (using results of Fig. 5); 2- adding that value to the experimentally obtained ΔATP₁.</p