Angiotensin II (AngII) is a peptide hormone that affects the cardiovascular system, not only through typical effects on the vasculature, kidneys, and heart, but also through less understood roles ...mediated by the brain and the immune system. Here, we address the hard-wired neural connections within the autonomic nervous system that modulate splenic immunity. Chronic AngII infusion triggers burst firing of the vagus nerve celiac efferent, an effect correlated with noradrenergic activation in the spleen and T cell egress. Bioelectronic stimulation of the celiac vagus nerve, in the absence of other challenges and independently from afferent signals to the brain, evokes the noradrenergic splenic pathway to promote release of a growth factor mediating neuroimmune crosstalk, placental growth factor (PlGF), and egress of CD8 effector T cells. Our findings also indicate that the neuroimmune interface mediated by PlGF and necessary for transducing the neural signal into an effective immune response is dependent on α-adrenergic receptor signaling.
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•Bioelectronic stimulation of celiac vagus nerve primes a splenic immune response•Vagus nerve stimulation selectively drives the egress of CD8+ effector T cells•Placental growth factor (PlGF) is a key mediator of the splenic neuroimmune coupling•Vagus nerve stimulation induces splenic PlGF through α-adrenergic receptors signaling
Carnevale et al. show that stimulation of the celiac vagus nerve primes an immune response in the spleen, driving selective egress of CD8+ T cells. This is mediated by placental growth factor, whose expression in the spleen is regulated by a α-adrenergic receptor signaling.
Abstract
Aims
Chronic increase of mineralocorticoids obtained by administration of deoxycorticosterone acetate (DOCA) results in salt-dependent hypertension in animals. Despite the lack of a ...generalized sympathoexcitation, DOCA-salt hypertension has been also associated to overdrive of peripheral nervous system in organs typically targeted by blood pressure (BP), as kidneys and vasculature. Aim of this study was to explore whether DOCA-salt recruits immune system by overactivating sympathetic nervous system in lymphoid organs and whether this is relevant for hypertension.
Methods and results
To evaluate the role of the neurosplenic sympathetic drive in DOCA-salt hypertension, we challenged splenectomized mice or mice with left coeliac ganglionectomy with DOCA-salt, observing that they were both unable to increase BP. Then, we evaluated by immunofluorescence and ELISA levels of the placental growth factor (PlGF) upon DOCA-salt challenge, which significantly increased the growth factor expression, but only in the presence of an intact neurosplenic sympathetic drive. When PlGF KO mice were subjected to DOCA-salt, they were significantly protected from the increased BP observed in WT mice under same experimental conditions. In addition, absence of PlGF hampered DOCA-salt mediated T cells co-stimulation and their consequent deployment towards kidneys where they infiltrated tissue and provoked end-organ damage.
Conclusion
Overall, our study demonstrates that DOCA-salt requires an intact sympathetic drive to the spleen for priming of immunity and consequent BP increase. The coupling of nervous system and immune cells activation in the splenic marginal zone is established through a sympathetic-mediated PlGF release, suggesting that this pathway could be a valid therapeutic target for hypertension.
Abstract only Multiple reports recognized that immunity participates in hypertension (HTN), although direct mechanisms that orchestrate the immune-vascular interaction are still unknown. Mice infused ...with AngiotensinII (AngII) typically increase blood pressure (BP), concomitantly recruiting immune cells in the vasculature, with CD8 T cells playing a crucial role. To investigate the molecular regulators of vascular-immune interface, we established a system of vessel organ culture, enabling the study of physiological properties of resistance arteries via a modified pressure myograph which keeps vessels alive and functional for several days, while co-culturing immune cells of interest. We tested potential direct effects of CD8 T cells isolated from AngII-HTN or vehicle mice on resistance arteries from naïve mice. After co-incubation in the organ culture, CD8 T cells from HTN mice significantly increased myogenic tone (MT) of arteries, while no effect was induced by CD8 T of vehicle mice (%MT=CD8 AngII :27±2; CD8 veh :16±2, p<0.01). Our previous studies showed that an intracellular signaling involved in the acquisition of CD8 effectors functions, phosphatidylinositol-3-kinase-γ (PI3Kγ), was also involved in AngII-induced HTN, being its inhibition protective toward increase in BP and in vascular resistance. Here we took advantage of a mouse model expressing constitutively active PI3Kγ (PI3Kγ CX ), to test immune functions of PI3Kγ in HTN. PI3Kγ CX showed a spontaneous hypertensive phenotype (SBP mmHg :129±1 vs WT:104±2, p<0.001) accompanied by infiltration of activated CD8 T cells in renal vasculature. Then, CD8 T cells isolated from PI3Kγ CX mice and placed in co-culture with WT arteries increased their MT. Conversely, the CD8 T cells from WT mice had no effect when cultured with vessels (%MT=CD8 CX :31±1; CD8 WT :19±1, p<0.001). To test the in vivo relevance, we performed an adoptive transfer of CD8 CX in WT mice, finding that they developed spontaneous HTN, while no effect was induced by CD8 WT (SBP mmHg : CD8 CX 128±2 vs CD8 WT 103±2, p<0.001). Taken together these data show that CD8 T cells from AngII-HTN mice are able to directly increase MT of resistance arteries and that PI3Kγ signaling is a crucial modulator of this effector function, likely contributing to BP increase.
Abstract only Obesity-related hypertension (HTN) is an epidemic health problem and a major risk factor for the development of cardiovascular disease. A consistent amount of research on the ...pathophysiological basis of obesity has implicated a crucial role of sympathetic overdrive. Interestingly, an overactivation of the sympathetic nervous system (SNS) accompanies HTN as well. Recent data highlighted that increased SNS activation in HTN is important in modulating immune responses, besides controlling typical cardiovascular functions. Whether neuroimmune mechanisms are relevant in obesity-induced HTN is still unknown. To test this hypothesis we subjected 6 week-old C57Bl/6J mice to high fat diet (HFD), as compared to low fat diet (LFD) and monitored blood pressure (BP), which started to rise after 2 months and steadily increased up to 4 months. We measured circulating noradrenalin, finding it significantly elevated 2 months after HFD. To gain insights in the potential SNS modulation of immunity, we measured by microneurography SNS activation on the splenic nerve. Firing frequency (FF, spikes in 10') of splenic nerve was increased in C57Bl/6J mice on HFD (FF:432±81) as compared to LFD (FF:86±10, p<0.01). Then, we surgically removed splenic innervation by left celiac ganglionectomy (CGX) and fed mice with HFD for 4 months. Despite CGX-mice become obese similarly to sham, as shown by body weight and microCT-measured fat pad, they were protected from HTN (SBP mmHg =CGX:105±4; sham:125±5, p<0.001), suggesting that the splenic sympathetic overdrive influences BP responses but not metabolic alterations. To look for molecular determinants, we analyzed the expression of Placental Growth Factor (PlGF), previously identified as a neuroimmune mediator, and found it significantly increased in the spleen upon HFD, but only with intact SNS innervation, since CGX-mice did not increase it. In the end, PlGF KO mice subjected to HFD, although becoming obese as much as WT, were protected from HTN (SBP mmHg =PlGF KO:103±4; WT:123±6, p<0.001). Interestingly, both CGX and PlGF KO mice displayed a reduced egression of activated T cells from the spleen upon HFD feeding, suggesting that the protection observed could be related to reduced infiltration of activated lymphocytes in target organs.
Abstract only Vascular endothelial growth factors (VEGFs) are mainly known as angiogenic factors but they also play less explored immune-modulating functions. In the past years we found that one of ...the VEGF family members, Placental Growth Factor (PlGF), is critical for hypertension. PlGF is recruited in the spleen by Angiotensin II (AngII) to prime T cell costimulation through the expression of CD86 in antigen presenting cells (APCs). Here we show that AngII also recruits VEGF-B, PlGF homolog, in the spleen and its selective splenic ablation, obtained by transplanting VEGF-B KO spleen in WT mice, protects from immune activation and hypertension. In the search for a downstream common mediator of PlGF/VEGF-B, we analyzed the splenic expression of VEGFR1, a major candidate receptor, which was activated by AngII. However, mice genetically defective of VEGFR1 signaling (Flt1-TK) become hypertensive as WT when subjected to AngII (SBP: Flt1-TK 139±2; WT 138±2; ***p< 0.001), thus ruling out its role. Since the expression of PlGF/VEGF-B was confined to the stromal and marginal zone (MZ), where lymphoid and innate zones are entangled with noradrenergic fibers, we analyzed Neuropilin1 (Nrp1), a secondary receptor for PlGF/VEGF-B, functioning in neuronal guidance and dendritic cell maturation. Immunofluorescence revealed that AngII increases Nrp1 expression and flow cytometry indicated a localization of the receptor on myeloid APCs. Splenic denervation reduced Nrp1 expression on APCs (from 29 ± 3% to 14 ± 2%), further supporting that it mediates PlGF/VEGF-B signaling. In the end, we selectively deleted the receptor in myeloid lineage, generating Nrp1 myeloidKO mice, thus blocking the effect of PlGF/VEGF-B on the APCs. The in vitro induction of DC maturation in monocytes isolated from Nrp1 myeloidKO mice failed to produce mature APC, thus indicating the requirement of this pathway for the initiation of the adaptive immune response. AngII fails to induce T cell costimulation and egression from the spleen, and ultimately increase BP (SBP: Nrp1 myeloidWT mice 140±3; Nrp1 myeloidKO mice 105±2; ***p< 0.001). Overall we demonstrate that PlGF/VEGF-B/Nrp1 is a critical neuroimmune mechanism responsible for initiation of the adaptive immune response in hypertension.
Abstract only In the search for mechanisms regulating the pathophysiological relationship existing between hypertension and immune system, CD8 effector T cells emerge as possible mediators of target ...organ colonization. However, how the crosstalk at vascular interfaces is established still remains unidentified. Phosphatidylinositol-3-kinase gamma (PI3Kγ) is an intracellular signaling involved in the acquisition of CD8 effectors functions and was previously showed by our group as involved in hypertension. The hypothesis here tested rely on the possibility that PI3Kγ could regulate trafficking of immune cells at vascular interfaces relevant for hypertension. By taking advantage of a knock-in mouse model, expressing a constitutively active PI3Kγ isoform (PI3Kγ CX/CX ), we conceived an experimental setting allowing to assess the direct crosstalk between immune cells and vasculature, obtained by co-culturing CD8 T cells with isolated resistance arteries mounted in pressurized systems. Thus, CD8 isolated from PI3Kγ CX/CX mice were co-cultured for 3 days with WT resistance arteries and, after the incubation period, vascular function was tested by challenging arteries to increasing intraluminal pressure to detect myogenic tone (MT). CD8 T cells with constitutively active PI3Kγ spontaneously increased MT of mesenteric arteries, a behavior typically associated with hypertension. Conversely, the same T cell population from WT mice, had no effect when cultured with vessels. In addition, PI3Kγ CX/CX CD8 infiltrated naïve vessels upon co-culture suggesting an intrinsic ability to colonize resistance districts relevant for hypertension. To test the in vivo relevance of the observed phenotype, we performed an adoptive transfer of PI3Kγ CX/CX CD8 in WT mice, finding that they developed spontaneous hypertension, accompanied by infiltration of effector CD8 in perivascular spaces of resistance districts as kidneys. Taken together these data show that PI3Kγ signaling in CD8 T cells is crucial for their effector functions in target vasculature of hypertension, likely contributing to BP increase by increasing peripheral resistance. These results also suggest translational potential as PI3Kγ inhibitors could modulate the immune response involved in hypertension.
Abstract only Obesity is an epidemic condition associated with several cardiovascular comorbidities, as hypertension. Although the molecular mechanisms related to obesity-induced hypertension are not ...well understood, the sympathetic nervous system (SNS) overactivation typically accompanying obesity has been ascribed as one of the main culprits. Thus far, mechanistic studies focused on the effects of sympathetic overdrive on peripheral resistances. Recent data showing that SNS activation also dictates immune responses involved in hypertension, made us hypothesize whether obesity induced by high fat diet (HFD) could recruit splenic SNS efferent. Thus we subjected WT mice to HFD, as compared to low fat diet (LFD), and monitored the hypertensive response. Blood pressure (BP) started to rise after two months, and hypertensive disease became clearly manifest within 4 months. To gain mechanistic insights responsible for BP increase, we assessed peripheral SNS activation at a time point preceding overt hypertension (i.e. 2 months after HFD). We found elevated circulating noradrenalin levels in HFD mice and, more important, a specific pattern of activated splenic sympathetic overdrive, as recorded by microneurography. WT mice subjected by surgical removal of the left celiac ganglion (CGX) where the splenic nerve originate, before receiving HFD, were protected from hypertension, despite developing obesity the same. The fact that splenectomized mice showed an overlapping response to HFD, supported the conclusion that the sympathetic-immune axis is crucial for HFD-induced hypertension but not for weight regulation. To look for molecular determinants, we analyzed the expression of Placental Growth Factor (PlGF), previously identified as a neuroimmune mediator of other hypertensive challenges. PlGF was significantly overexpressed in the spleen upon HFD, but only when SNS innervation was intact, as CGX mice failed to induce its expression. In the end, PlGF KO mice subjected to HFD, although becoming obese as much as WT did, were protected from hypertension. Overall our results demonstrate that a splenic neuroimmune drive is crucial to allow the onset of hypertensive disease associated with obesity, although having no effect in body weight increase in response to HFD.
OBJECTIVE—EMILIN-1 (elastin microfibrils interface located protein-1) protein inhibits pro–TGF-β (transforming growth factor-β) proteolysis and limits TGF-β bioavailability in vascular extracellular ...matrix. Emilin1 null mice display increased vascular TGF-β signaling and are hypertensive. Because EMILIN-1 is expressed in vessels from embryonic life to adulthood, we aimed at unravelling whether the hypertensive phenotype of Emilin1 null mice results from a developmental defect or lack of homeostatic role in the adult.
APPROACH AND RESULTS—By using a conditional gene targeting inactivating EMILIN-1 in smooth muscle cells of adult mice, we show that increased blood pressure in mice with selective smooth muscle cell ablation of EMILIN-1 depends on enhanced myogenic tone. Mechanistically, we unveil that higher TGF-β signaling in smooth muscle cells stimulates HB-EGF (heparin-binding epidermal growth factor) expression and subsequent transactivation of EGFR (epidermal growth factor receptor). With increasing intraluminal pressure in resistance arteries, the cross talk established by TGF-β and EGFR signals recruits TRPC6 (TRP transient receptor potential classical type 6) and TRPM4 (TRP melastatin type 4) channels, lastly stimulating voltage-dependent calcium channels and potentiating myogenic tone. We found reduced EMILIN-1 and enhanced myogenic tone, dependent on increased TGF-β-EGFR signaling, in resistance arteries from hypertensive patients.
CONCLUSIONS—Taken together, our findings implicate an unexpected role of the TGF-β-EGFR pathway in hypertension with current translational perspectives.
Among interleukin-6 inhibitors suggested for use in COVID-19, there are few robust evidences for the efficacy of sarilumab. Herein, we evaluated the efficacy and safety of sarilumab in severe ...COVID-19.
In this phase 3, open-labeled, randomized clinical trial, conducted at 5 Italian hospitals, adults with severe COVID-19 pneumonia (excluding mechanically ventilated) were randomized 2:1 to receive intravenous sarilumab (400 mg, repeatable after 12 h) plus standard of care (SOC) (arm A) or to continue SOC (arm B). Randomization was web-based. As post-hoc analyses, the participants were stratified according to baseline inflammatory parameters. The primary endpoint was analysed on the modified Intention-To-Treat population, including all the randomized patients who received any study treatment (sarilumab or SOC). It was time to clinical improvement of 2 points on a 7-points ordinal scale, from baseline to day 30. We used Kaplan Meier method and log-rank test to compare the primary outcome between two arms, and Cox regression stratified by clinical center and adjusted for severity of illness, to estimate the hazard ratio (HR). The trial was registered with EudraCT (2020-001390-76).
Between May 2020 and May 2021, 191 patients were assessed for eligibility, of whom, excluding nine dropouts, 176 were assigned to arm A (121) and B (55). At day 30, no significant differences in the primary endpoint were found (88% 95% CI 81–94 in arm A vs 85% 74–93, HR 1.07 0.8–1.5 in arm B; log-rank p = 0.50). After stratifying for inflammatory parameters, arm A showed higher probability of improvement than B without statistical significance in the strata with C reactive protein (CRP) < 7 mg/dL (88% 77–96 vs 79% 63–91, HR 1.55 0.9–2.6; log-rank p = 0.049) and in the strata with lymphocytes <870/mmc (90% 79–96) vs (73% 55–89, HR 1.53 0.9–2.7; log-rank p = 0.058). Overall, 39/121 (32%) AEs were reported in arm A and 14/55 (23%) in B (p = 0.195), while serious AEs were 22/121 (18%) and 7/55 (11%), respectively (p = 0.244). There were no treatment-related deaths.
The efficacy of sarilumab in severe COVID-19 was not demonstrated both in the overall and in the stratified for severity analysis population. Exploratory analyses suggested that subsets of patients with lower CRP values or lower lymphocyte counts might have had benefit with sarilumab treatment, but this finding would require replication in other studies. The relatively low rate of concomitant corticosteroid use, could partially explain our results.
This study was supported by INMI “Lazzaro Spallanzani” Ricerca Corrente Linea 1 on emerging and reemerging infections, funded by Italian Ministry of Health.
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