RESUMEN
Benznidazole (Bzl), the drug of choice in many countries for the treatment of Chagas disease, leads to parasite clearance in the early stages of infection and contributes to immunomodulation. In addition to its parasiticidal effect, Bzl inhibits the NF-κB pathway. In this regard, we have previously described that this occurs through IL-10/STAT3/SOCS3 pathway. PI3K pathway is involved in the regulation of the immune system by inhibiting NF-κB pathway through STAT3. In this work, the participation of PI3K in the immunomodulatory effects of Bzl in cardiac and immune cells, the main targets of Chagas disease, was further studied. For that, we use a murine primary cardiomyocyte culture and a monocyte/macrophage cell line (RAW 264.7), stimulated with LPS in presence of LY294002, an inhibitor of PI3K. Under these conditions, Bzl could neither increase SOCS3 expression nor inhibit the NOS2 mRNA expression and the release of NOx, both in cardiomyocytes and macrophages. Macrophages are crucial in the development of Chronic Chagas Cardiomyopathy. Thus, to deepen our understanding of how Bzl acts, the expression profile of M1-M2 macrophage markers was evaluated. Bzl inhibited the release of NOx (M1 marker) and increased the expression of Arginase I (M2 marker) and a negative correlation was found between them. Besides, LPS increased the expression of pro-inflammatory cytokines. Bzl treatment not only inhibited this effect but also increased the expression of typical M2-macrophage markers like Mannose Receptor, TGF-ß, and VEGF-A. Moreover, Bzl increased the expression of PPAR-γ and PPAR-α, known as key regulators of macrophage polarization. PI3K directly regulates M1-to-M2 macrophage polarization. Since p110δ, catalytic subunit of PI3Kδ, is highly expressed in immune cells, experiments were carried out in presence of CAL-101, a specific inhibitor of this subunit. Under this condition, Bzl could neither increase SOCS3 expression nor inhibit NF-κB pathway. Moreover, Bzl not only failed to inhibit the expression of pro-inflammatory cytokines (M1 markers) but also could not increase M2 markers. Taken together these results demonstrate, for the first time, that the anti-inflammatory effect of Bzl depends on PI3K activity in a cell line of murine macrophages and in primary culture of neonatal cardiomyocytes. Furthermore, Bzl-mediated increase expression of M2-macrophage markers involves the participation of the p110δ catalytic subunit of PI3Kδ.
Asunto(s)
Antiinflamatorios/farmacología , Cardiomiopatía Chagásica/tratamiento farmacológico , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Nitroimidazoles/farmacología , Animales , Animales Recién Nacidos , Antiinflamatorios/uso terapéutico , Cardiomiopatía Chagásica/inmunología , Cromonas/farmacología , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Modelos Animales de Enfermedad , Femenino , Humanos , Lipopolisacáridos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Morfolinas/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/inmunología , Miocitos Cardíacos/metabolismo , Nitroimidazoles/uso terapéutico , Cultivo Primario de Células , Células RAW 264.7RESUMEN
Intolerance is the most common reason for kinase inhibitor (KI) discontinuation in chronic lymphocytic leukemia (CLL). Umbralisib, a novel highly selective phosphatidylinositol 3-kinase δ (PI3Kδ)/CK1ε inhibitor, is active and well tolerated in CLL patients. In this phase 2 trial (NCT02742090), umbralisib was initiated at 800 mg/d in CLL patients requiring therapy, who were intolerant to prior BTK inhibitor (BTKi) or PI3K inhibitor (PI3Ki) therapy, until progression or toxicity. Primary end point was progression-free survival (PFS). Secondary end points included time to treatment failure and safety. DNA was genotyped for CYP3A4, CYP3A5, and CYP2D6 polymorphisms. Fifty-one patients were enrolled (44 BTKi intolerant and 7 PI3Kδi intolerant); median age was 70 years (range, 48-96), with a median of 2 prior lines of therapy (range, 1-7), 24% had del17p and/or TP53 mutation, and 65% had unmutated IGHV. Most common adverse events (AEs) leading to prior KI discontinuation were rash (27%), arthralgia (18%), and atrial fibrillation (16%). Median PFS was 23.5 months (95% CI, 13.1-not estimable), with 58% of patients on umbralisib for a longer duration than prior KI. Most common (≥5%) grade ≥3 AEs on umbralisib (all causality) were neutropenia (18%), leukocytosis (14%), thrombocytopenia (12%), pneumonia (12%), and diarrhea (8%). Six patients (12%) discontinued umbralisib because of an AE. Eight patients (16%) had dose reductions and were successfully rechallenged. These are the first prospective data to confirm that switching from a BTKi or alternate PI3Ki to umbralisib in this BTKi- and PI3Ki-intolerant CLL population can result in durable well-tolerated responses.
Asunto(s)
Antineoplásicos/uso terapéutico , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Adenina/efectos adversos , Adenina/análogos & derivados , Adenina/uso terapéutico , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Anciano , Anciano de 80 o más Años , Antineoplásicos/efectos adversos , Enfermedades Cardiovasculares/inducido químicamente , Erupciones por Medicamentos/etiología , Resistencia a Antineoplásicos , Femenino , Enfermedades Gastrointestinales/inducido químicamente , Compuestos Heterocíclicos de 4 o más Anillos/efectos adversos , Humanos , Estimación de Kaplan-Meier , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/mortalidad , Masculino , Persona de Mediana Edad , Piperidinas/efectos adversos , Piperidinas/uso terapéutico , Supervivencia sin Progresión , Inhibidores de Proteínas Quinasas/efectos adversosRESUMEN
Upon in vitro stimulation, neutrophils undergo a cell death named netosis. This process is characterized by extracellular release of chromatin scaffold associated with granular and cytoplasmic proteins, which together, ensnare and kill microbes. We have previously described that interaction of Leishmania amazonensis with human neutrophils leads to the release of neutrophil extracellular traps, which trap and kill the parasite. However, the signaling leading to Leishmania induced netosis is still unknown. Thus, we sought to evaluate signaling events that drive L. amazonensis induced neutrophil extracellular trap release from human neutrophils. Here, we found that PI3K, independently of protein kinase B, has a role in parasite-induced netosis. We also described that the main isoforms involved are PI3Kγ and PI3Kδ, which work in reactive oxygen species-dependent and -independent ways, respectively. We demonstrated that activation of ERK downstream of PI3Kγ is important to trigger reactive oxygen species-dependent, parasite-induced netosis. Pharmacological inhibition of protein kinase C also significantly decreased parasite-induced neutrophil extracellular trap release. Intracellular calcium, regulated by PI3Kδ, represents an alternative reactive oxygen species-independent pathway of netosis stimulated by L. amazonensis Finally, intracellular calcium mobilization and reactive oxygen species generation are the major regulators of parasite-induced netosis. Our results contribute to a better understanding of the signaling behind netosis induced by interactions between Leishmania and neutrophils.