Dual endothelin antagonist aprocitentan for resistant hypertension (PRECISION): a multicentre, blinded, randomised, parallel-group, phase 3 trial.

BACKGROUND: Resistant hypertension is associated with increased cardiovascular risk. The endothelin pathway has been implicated in the pathogenesis of hypertension, but it is currently not targeted therapeutically, thereby leaving this relevant pathophysiological pathway unopposed with currently available drugs. The aim of the study was to assess the blood pressure lowering efficacy of the dual endothelin antagonist aprocitentan in patients with resistant hypertension. METHODS: PRECISION was a multicentre, blinded, randomised, parallel-group, phase 3 study, which was done in hospitals or research centres in Europe, North America, Asia, and Australia. Patients were eligible for randomisation if their sitting systolic blood pressure was 140 mm Hg or higher despite taking standardised background therapy consisting of three antihypertensive drugs, including a diuretic. The study consisted of three sequential parts: part 1 was the 4-week double-blind, randomised, and placebo-controlled part, in which patients received aprocitentan 12·5 mg, aprocitentan 25 mg, or placebo in a 1:1:1 ratio; part 2 was a 32-week single (patient)-blind part, in which all patients received aprocitentan 25 mg; and part 3 was a 12-week double-blind, randomised, and placebo-controlled withdrawal part, in which patients were re-randomised to aprocitentan 25 mg or placebo in a 1:1 ratio. The primary and key secondary endpoints were changes in unattended office systolic blood pressure from baseline to week 4 and from withdrawal baseline to week 40, respectively. Secondary endpoints included 24-h ambulatory blood pressure changes. The study is registered on ClinicalTrials.gov, NCT03541174. FINDINGS: The PRECISION study was done from June 18, 2018, to April 25, 2022. 1965 individuals were screened and 730 were randomly assigned. Of these 730 patients, 704 (96%) completed part 1 of the study; of these, 613 (87%) completed part 2 and, of these, 577 (94%) completed part 3 of the study. The least square mean (SE) change in office systolic blood pressure at 4 weeks was -15·3 (SE 0·9) mm Hg for aprocitentan 12·5 mg, -15·2 (0·9) mm Hg for aprocitentan 25 mg, and -11·5 (0·9) mm Hg for placebo, for a difference versus placebo of -3·8 (1·3) mm Hg (97·5% CI -6·8 to -0·8, p=0·0042) and -3·7 (1·3) mm Hg (-6·7 to -0·8; p=0·0046), respectively. The respective difference for 24 h ambulatory systolic blood pressure was -4·2 mm Hg (95% CI -6·2 to -2·1) and -5·9 mm Hg (-7·9 to -3·8). After 4 weeks of withdrawal, office systolic blood pressure significantly increased with placebo versus aprocitentan (5·8 mm Hg, 95% CI 3·7 to 7·9, p<0·0001). The most frequent adverse event was mild-to-moderate oedema or fluid retention, occurring in 9%, 18%, and 2% for patients receiving aprocitentan 12·5 mg, 25 mg, and placebo, during the 4-week double-blind part, respectively. This event led to discontinuation in seven patients treated with aprocitentan. During the trial, a total of 11 treatment-emergent deaths occurred, none of which were regarded by the investigators to be related to study treatment. INTERPRETATION: In patients with resistant hypertension, aprocitentan was well tolerated and superior to placebo in lowering blood pressure at week 4 with a sustained effect at week 40. FUNDING: Idorsia Pharmaceuticals and Janssen Biotech.

The Inflammasome Drives GSDMD-Independent Secondary Pyroptosis and IL-1 Release in the Absence of Caspase-1 Protease Activity.

Inflammasomes activate the protease caspase-1, which cleaves interleukin-1ß and interleukin-18 to generate the mature cytokines and controls their secretion and a form of inflammatory cell death called pyroptosis. By generating mice expressing enzymatically inactive caspase-1C284A, we provide genetic evidence that caspase-1 protease activity is required for canonical IL-1 secretion, pyroptosis, and inflammasome-mediated immunity. In caspase-1-deficient cells, caspase-8 can be activated at the inflammasome. Using mice either lacking the pyroptosis effector gasdermin D (GSDMD) or expressing caspase-1C284A, we found that GSDMD-dependent pyroptosis prevented caspase-8 activation at the inflammasome. In the absence of GSDMD-dependent pyroptosis, the inflammasome engaged a delayed, alternative form of lytic cell death that was accompanied by the release of large amounts of mature IL-1 and contributed to host protection. Features of this cell death modality distinguished it from apoptosis, suggesting it may represent a distinct form of pro-inflammatory regulated necrosis.

Francisella requires dynamic type VI secretion system and ClpB to deliver effectors for phagosomal escape.

Francisella tularensis is an intracellular pathogen that causes the fatal zoonotic disease tularaemia. Critical for its pathogenesis is the ability of the phagocytosed bacteria to escape into the cell cytosol. For this, the bacteria use a non-canonical type VI secretion system (T6SS) encoded on the Francisella pathogenicity island (FPI). Here we show that in F. novicida T6SS assembly initiates at the bacterial poles both in vitro and within infected macrophages. T6SS dynamics and function depends on the general purpose ClpB unfoldase, which specifically colocalizes with contracted sheaths and is required for their disassembly. T6SS assembly depends on iglF, iglG, iglI and iglJ, whereas pdpC, pdpD, pdpE and anmK are dispensable. Importantly, strains lacking pdpC and pdpD are unable to escape from phagosome, activate AIM2 inflammasome or cause disease in mice. This suggests that PdpC and PdpD are T6SS effectors involved in phagosome rupture.

Guanylate-binding proteins promote activation of the AIM2 inflammasome during infection with Francisella novicida.

The AIM2 inflammasome detects double-stranded DNA in the cytosol and induces caspase-1-dependent pyroptosis as well as release of the inflammatory cytokines interleukin 1ß (IL-1ß) and IL-18. AIM2 is critical for host defense against DNA viruses and bacteria that replicate in the cytosol, such as Francisella tularensis subspecies novicida (F. novicida). The activation of AIM2 by F. novicida requires bacteriolysis, yet whether this process is accidental or is a host-driven immunological mechanism has remained unclear. By screening nearly 500 interferon-stimulated genes (ISGs) through the use of small interfering RNA (siRNA), we identified guanylate-binding proteins GBP2 and GBP5 as key activators of AIM2 during infection with F. novicida. We confirmed their prominent role in vitro and in a mouse model of tularemia. Mechanistically, these two GBPs targeted cytosolic F. novicida and promoted bacteriolysis. Thus, in addition to their role in host defense against vacuolar pathogens, GBPs also facilitate the presentation of ligands by directly attacking cytosolic bacteria.

Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases.

Lipopolysaccharide from Gram-negative bacteria is sensed in the host cell cytoplasm by a non-canonical inflammasome pathway that ultimately results in caspase-11 activation and cell death. In mouse macrophages, activation of this pathway requires the production of type-I interferons, indicating that interferon-induced genes have a critical role in initiating this pathway. Here we report that a cluster of small interferon-inducible GTPases, the so-called guanylate-binding proteins, is required for the full activity of the non-canonical caspase-11 inflammasome during infections with vacuolar Gram-negative bacteria. We show that guanylate-binding proteins are recruited to intracellular bacterial pathogens and are necessary to induce the lysis of the pathogen-containing vacuole. Lysis of the vacuole releases bacteria into the cytosol, thus allowing the detection of their lipopolysaccharide by a yet unknown lipopolysaccharide sensor. Moreover, recognition of the lysed vacuole by the danger sensor galectin-8 initiates the uptake of bacteria into autophagosomes, which results in a reduction of caspase-11 activation. These results indicate that host-mediated lysis of pathogen-containing vacuoles is an essential immune function and is necessary for efficient recognition of pathogens by inflammasome complexes in the cytosol.

Systems-level overview of host protein phosphorylation during Shigella flexneri infection revealed by phosphoproteomics.

The enteroinvasive bacterium Shigella flexneri invades the intestinal epithelium of humans. During infection, several injected effector proteins promote bacterial internalization, and interfere with multiple host cell responses. To obtain a systems-level overview of host signaling during infection, we analyzed the global dynamics of protein phosphorylation by liquid chromatography-tandem MS and identified several hundred of proteins undergoing a phosphorylation change during the first hours of infection. Functional bioinformatic analysis revealed that they were mostly related to the cytoskeleton, transcription, signal transduction, and cell cycle. Fuzzy c-means clustering identified six temporal profiles of phosphorylation and a functional module composed of ATM-phosphorylated proteins related to genotoxic stress. Pathway enrichment analysis defined mTOR as the most overrepresented pathway. We showed that mTOR complex 1 and 2 were required for S6 kinase and AKT activation, respectively. Comparison with a published phosphoproteome of Salmonella typhimurium-infected cells revealed a large subset of coregulated phosphoproteins. Finally, we showed that S. flexneri effector OspF affected the phosphorylation of several hundred proteins, thereby demonstrating the wide-reaching impact of a single bacterial effector on the host signaling network.