Bone marrow megakaryocytes engulf neutrophils in a phenomenon termed emperipolesis. We show here that emperipolesis is a dynamic process mediated actively by both lineages, in part through the β2-integrin/ICAM-1/ezrin pathway. Tethered neutrophils enter in membrane-bound vesicles before penetrating into the megakaryocyte cytoplasm. Intracytoplasmic neutrophils develop membrane contiguity with the demarcation membrane system, thereby transferring membrane to the megakaryocyte and to daughter platelets. This phenomenon occurs in otherwise unmanipulated murine marrow in vivo, resulting in circulating platelets that bear membrane from non-megakaryocytic hematopoietic donors. Transit through megakaryocytes can be completed as rapidly as minutes, after which neutrophils egress intact. Emperipolesis is amplified in models of murine inflammation associated with platelet overproduction, contributing to platelet production in vitro and in vivo. These findings identify emperipolesis as a new cell-in-cell interaction that enables neutrophils and potentially other cells passing through the megakaryocyte cytoplasm to modulate the production and membrane content of platelets.

Objective Human TNKS, encoding tankyrase 1 (TNKS1), localizes to a susceptibility locus for obesity and type 2 diabetes mellitus (T2DM). Here, we addressed the therapeutic potential of G007-LK, a TNKS-specific inhibitor, for obesity and T2DM. Methods We administered G007-LK to diabetic db/db mice and measured the impact on body weight, abdominal adiposity, and serum metabolites. Muscle, liver, and white adipose tissues were analyzed by quantitative RT-PCR and western blotting to determine TNKS inhibition, lipolysis, beiging, adiponectin level, mitochondrial oxidative metabolism and mass, and gluconeogenesis. Protein interaction and PARylation analyses were carried out by immunoprecipitation, pull-down and in situ proximity ligation assays. Results TNKS inhibition reduced body weight gain, abdominal fat content, serum cholesterol levels, steatosis, and proteins associated with lipolysis in diabetic db/db mice. We discovered that TNKS associates with PGC-1α and that TNKS inhibition attenuates PARylation of PGC-1α, contributing to increased PGC-1α level in WAT and muscle in db/db mice. PGC-1α upregulation apparently modulated transcriptional reprogramming to increase mitochondrial mass and fatty acid oxidative metabolism in muscle, beiging of WAT, and raised circulating adiponectin level in db/db mice. This was in sharp contrast to the liver, where TNKS inhibition in db/db mice had no effect on PGC-1α expression, lipid metabolism, or gluconeogenesis. Conclusion Our study unravels a novel molecular mechanism whereby pharmacological inhibition of TNKS in obesity and diabetes enhances oxidative metabolism and ameliorates lipid disorder. This happens via tissue-specific PGC-1α-driven transcriptional reprogramming in muscle and WAT, without affecting liver. This highlights inhibition of TNKS as a potential pharmacotherapy for obesity and T2DM.

Aims/hypothesis Chronic low-grade inflammation with local upregulation of proinflammatory molecules plays a role in the progression of obesity-related renal injury. Reduced serum concentration of anti-inflammatory adiponectin may promote chronic inflammation. Here, we investigated the potential anti-inflammatory and renoprotective effects and mechanisms of action of AdipoRon, an adiponectin receptor agonist. Methods Wild-type DBA/2J mice were fed with high-fat diet (HFD) supplemented or not with AdipoRon to model obesity-induced metabolic endotoxaemia and chronic low-grade inflammation and we assessed changes in the glomerular morphology and expression of proinflammatory markers. We also treated human glomeruli ex vivo and human podocytes in vitro with AdipoRon and bacterial lipopolysaccharide (LPS), an endotoxin upregulated in obesity and diabetes, and analysed the secretion of inflammatory cytokines, activation of inflammatory signal transduction pathways, apoptosis and migration. Results In HFD-fed mice, AdipoRon attenuated renal inflammation, as demonstrated by reduced expression of glomerular activated NF-κB p65 subunit (NF-κB-p65) (70%, p < 0.001), TNFα (48%, p < 0.01), IL-1β (51%, p < 0.001) and TGFβ (46%, p < 0.001), renal IL-6 and IL-4 (21% and 20%, p < 0.05), and lowered glomerular F4/80-positive macrophage infiltration (31%, p < 0.001). In addition, AdipoRon ameliorated HFD-induced glomerular hypertrophy (12%, p < 0.001), fibronectin accumulation (50%, p < 0.01) and podocyte loss (12%, p < 0.001), and reduced podocyte foot process effacement (15%, p < 0.001) and thickening of the glomerular basement membrane (18%, p < 0.001). In cultured podocytes, AdipoRon attenuated the LPS-induced activation of the central inflammatory signalling pathways NF-κB-p65, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38-MAPK) (30%, 36% and 22%, respectively, p < 0.001), reduced the secretion of TNFα (32%, p < 0.01), and protected against podocyte apoptosis and migration. In human glomeruli ex vivo, AdipoRon reduced the LPS-induced secretion of inflammatory cytokines IL-1β, IL-18, IL-6 and IL-10. Conclusions/interpretation AdipoRon attenuated the renal expression of proinflammatory cytokines in HFD-fed mice and LPS-stimulated human glomeruli, which apparently contributed to the amelioration of glomerular inflammation and injury. Mechanistically, based on assays on cultured podocytes, AdipoRon reduced LPS-induced activation of the NF-κB-p65, JNK and p38-MAPK pathways, thereby impelling the decrease in apoptosis, migration and secretion of TNFα. We conclude that the activation of the adiponectin receptor by AdipoRon is a potent strategy to attenuate endotoxaemia-associated renal inflammation. Graphical abstract

Bone marrow megakaryocytes engulf neutrophils in a phenomenon termed emperipolesis. We show here that emperipolesis is a dynamic process mediated actively by both lineages, in part through the β2-integrin/ICAM-1/ezrin pathway. Tethered neutrophils enter in membrane-bound vesicles before penetrating into the megakaryocyte cytoplasm. Intracytoplasmic neutrophils develop membrane contiguity with the demarcation membrane system, thereby transferring membrane to the megakaryocyte and to daughter platelets. This phenomenon occurs in otherwise unmanipulated marrow in vivo, resulting in circulating platelets that bear membrane from non-megakaryocytic hematopoietic donors. Transit through megakaryocytes can be completed as rapidly as minutes, after which neutrophils egress intact. Emperipolesis is amplified in models of inflammation associated with platelet overproduction, contributing to platelet production in vitro and in vivo. These findings identify emperipolesis as a new cell-in-cell interaction that enables neutrophils and potentially other cells passing through the megakaryocyte cytoplasm to modulate the production and membrane content of platelets.