Comparing pulsed field electroporation and radiofrequency ablation for the treatment of paroxysmal atrial fibrillation: design and rationale of the BEAT PAROX-AF randomized clinical trial.

AIMS: Using thermal-based energy sources [radiofrequency (RF) energy/cryo energy] for catheter ablation is considered effective and safe when performing pulmonary vein isolation (PVI) in patients with paroxysmal atrial fibrillation (AF). However, treatment success remains limited and complications can occur due to the propagation of thermal energy into non-target tissues. We aim to compare pulsed field ablation (PFA) with RF ablation in terms of efficacy and safety for patients with drug-resistant paroxysmal AF. METHODS AND RESULTS: The BEAT PAROX-AF trial is a European multicentre, superiority, open-label randomized clinical trial in two parallel groups. A total of 292 participants were recruited in 9 high-volume European clinical centres in 5 countries. Patients with paroxysmal AF were randomized to PFA (FARAPULSE Endocardial Ablation System©, Boston Scientific) or RF using the CLOSE protocol with contact force sensing catheter (SmartTouch© catheter and CARTO© Biosense Webster). The primary endpoint will be the 1-year recurrence of atrial arrhythmia, and the major secondary safety endpoint will be the occurrence of acute (<7 days) procedure-related serious adverse events, or pulmonary vein stenosis, or atrio-oesophageal fistula up to 12 months. Additionally, five sub-studies investigate the effect of PFA on oesophageal safety, cerebral lesions, cardiac autonomic nervous system, durability of PVI as assessed during redo ablation procedures, and atrial and ventricular function. The study began on 27 December 2021 and concluded recruitment on 17 January 2024. Results will be available in mid-2025. CONCLUSION: The BEAT PAROX-AF trial aims to provide critical insights into the optimal treatment approach for patients with paroxysmal AF.

HCV glycoprotein E2 is a novel BDCA-2 ligand and acts as an inhibitor of IFN production by plasmacytoid dendritic cells.

The elimination of hepatitis C virus (HCV) in > 50% of chronically infected patients by treatment with IFN-α suggests that plasmacytoid dendritic cells (pDCs), major producers of IFN-α, play an important role in the control of HCV infection. However, despite large amounts of Toll-like receptor 7-mediated IFN-α, produced by pDCs exposed to HCV-infected hepatocytes, HCV still replicates in infected liver. Here we show that HCV envelope glycoprotein E2 is a novel ligand of pDC C-type lectin immunoreceptors (CLRs), blood DC antigen 2 (BDCA-2) and DC-immunoreceptor (DCIR). HCV particles inhibit, via binding of E2 glycoprotein to CLRs, production of IFN-α and IFN-λ in pDCs exposed to HCV-infected hepatocytes, and induce in pDCs a rapid phosphorylation of Akt and Erk1/2, in a manner similar to the crosslinking of BDCA-2 or DCIR. Blocking of BDCA-2 and DCIR with Fab fragments of monoclonal antibodies preserves the capacity of pDCs to produce type I and III IFNs in the presence of HCV particles. Thus, negative interference of CLR signaling triggered by cell-free HCV particles with Toll-like receptor signaling triggered by cell-associated HCV results in the inhibition of the principal pDC function, production of IFN.

Hepatitis C virus fails to activate NF-κB signaling in plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) respond to viral infection by production of alpha interferon (IFN-α), proinflammatory cytokines, and cell differentiation. The elimination of hepatitis C virus (HCV) in more than 50% of chronically infected patients by treatment with IFN-α suggests that pDCs can play an important role in the control of HCV infection. pDCs exposed to HCV-infected hepatoma cells, in contrast to cell-free HCV virions, produce large amounts of IFN-α. To further investigate the molecular mechanism of HCV sensing, we studied whether exposure of pDCs to HCV-infected hepatoma cells activates, in parallel to interferon regulatory factor 7 (IRF7)-mediated production of IFN-α, nuclear factor kappa B (NF-κB)-dependent pDC responses, such as expression of the differentiation markers CD40, CCR7, CD86, and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and secretion of the proinflammatory cytokines TNF-α and interleukin 6 (IL-6). We demonstrate that exposure of pDCs to HCV-infected hepatoma cells surprisingly did not induce phosphorylation of NF-κB or cell surface expression of CD40, CCR7, CD86, or TRAIL or secretion of TNF-α and IL-6. In contrast, CpG-A and CpG-B induced production of TNF-α and IL-6 in pDCs exposed to the HCV-infected hepatoma cells, showing that cell-associated virus did not actively inhibit Toll-like receptor (TLR)-mediated NF-κB phosphorylation. Our results suggest that cell-associated HCV signals in pDCs via an endocytosis-dependent mechanism and IRF7 but not via the NF-κB pathway. In spite of IFN-α induction, cell-associated HCV does not induce a full functional response of pDCs. These findings contribute to the understanding of evasion of immune responses by HCV.

The MEK1/2-ERK Pathway Inhibits Type I IFN Production in Plasmacytoid Dendritic Cells.

Recent studies have reported that the crosslinking of regulatory receptors (RRs), such as blood dendritic cell antigen 2 (BDCA-2) (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses the production of type I interferons (IFN-I, α/ß/ω) and other cytokines in response to toll-like receptor 7 and 9 (TLR7/9) ligands. The exact mechanism of how this B cell receptor (BCR)-like signaling blocks TLR7/9-mediated IFN-I production is unknown. Here, we stimulated BCR-like signaling by ligation of RRs with BDCA-2 and ILT7 mAbs, hepatitis C virus particles, or BST2 expressing cells. We compared BCR-like signaling in proliferating pDC cell line GEN2.2 and in primary pDCs from healthy donors, and addressed the question of whether pharmacological targeting of BCR-like signaling can antagonize RR-induced pDC inhibition. To this end, we tested the TLR9-mediated production of IFN-I and proinflammatory cytokines in pDCs exposed to a panel of inhibitors of signaling molecules involved in BCR-like, MAPK, NF-ĸB, and calcium signaling pathways. We found that MEK1/2 inhibitors, PD0325901 and U0126 potentiated TLR9-mediated production of IFN-I in GEN2.2 cells. More importantly, MEK1/2 inhibitors significantly increased the TLR9-mediated IFN-I production blocked in both GEN2.2 cells and primary pDCs upon stimulation of BCR-like or phorbol 12-myristate 13-acetate-induced protein kinase C (PKC) signaling. Triggering of BCR-like and PKC signaling in pDCs resulted in an upregulation of the expression and phoshorylation of c-FOS, a downstream gene product of the MEK1/2-ERK pathway. We found that the total level of c-FOS was higher in proliferating GEN2.2 cells than in the resting primary pDCs. The PD0325901-facilitated restoration of the TLR9-mediated IFN-I production correlated with the abrogation of MEK1/2-ERK-c-FOS signaling. These results indicate that the MEK1/2-ERK pathway inhibits TLR9-mediated type I IFN production in pDCs and that pharmacological targeting of MEK1/2-ERK signaling could be a strategy to overcome immunotolerance of pDCs and re-establish their immunogenic activity.

Dual Role of the Tyrosine Kinase Syk in Regulation of Toll-Like Receptor Signaling in Plasmacytoid Dendritic Cells.

Crosslinking of regulatory immunoreceptors (RR), such as BDCA-2 (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses production of type-I interferon (IFN)-α/ß and other cytokines in response to Toll-like receptor (TLR) 7/9 ligands. This cytokine-inhibitory pathway is mediated by spleen tyrosine kinase (Syk) associated with the ITAM-containing adapter of RR. Here we demonstrate by pharmacological targeting of Syk that in addition to the negative regulation of TLR7/9 signaling via RR, Syk also positively regulates the TLR7/9 pathway in human pDCs. Novel highly specific Syk inhibitor AB8779 suppressed IFN-α, TNF-α and IL-6 production induced by TLR7/9 agonists in primary pDCs and in the pDC cell line GEN2.2. Triggering of TLR9 or RR signaling induced a differential kinetics of phosphorylation at Y352 and Y525/526 of Syk and a differential sensitivity to AB8779. Consistent with the different roles of Syk in TLR7/9 and RR signaling, a concentration of AB8779 insufficient to block TLR7/9 signaling still released the block of IFN-α production triggered via the RR pathway, including that induced by hepatitis B and C viruses. Thus, pharmacological targeting of Syk partially restored the main pDC function-IFN-α production. Opposing roles of Syk in TLR7/9 and RR pathways may regulate the innate immune response to weaken inflammation reaction.