Diabetes mellitus (DM) encompasses a multitude of secondary disorders, including heart disease. One of the most frequent and potentially life threatening disorders of DM-induced heart disease is ventricular tachycardia (VT). Here we show that toll-like receptor 2 (TLR2) and NLRP3 inflammasome activation in cardiac macrophages mediate the production of IL-1β in DM mice. IL-1β causes prolongation of the action potential duration, induces a decrease in potassium current and an increase in calcium sparks in cardiomyocytes, which are changes that underlie arrhythmia propensity. IL-1β-induced spontaneous contractile events are associated with CaMKII oxidation and phosphorylation. We further show that DM-induced arrhythmias can be successfully treated by inhibiting the IL-1β axis with either IL-1 receptor antagonist or by inhibiting the NLRP3 inflammasome. Our results establish IL-1β as an inflammatory connection between metabolic dysfunction and arrhythmias in DM.

Hemolytic diseases include a variety of conditions with diverse etiologies in which red blood cells are destroyed and large amounts of hemeproteins are released. Heme has been described as a potent proinflammatory molecule that is able to induce multiple innate immune responses, such as those triggered by TLR4 and the NLRP3 inflammasome, as well as necroptosis in macrophages. The mechanisms by which eukaryotic cells respond to the toxic effects induced by heme to maintain homeostasis are not fully understood, however. Here we describe a previously uncharacterized cellular response induced by heme: the formation of p62/SQTM1 aggregates containing ubiquitinated proteins in structures known as aggresome-like induced structures (ALIS). This action is part of a response driven by the transcription factor NRF2 to the excessive generation of reactive oxygen species induced by heme that results in the expression of genes involved in antioxidant responses, including p62/SQTM1. Furthermore, we show that heme degradation by HO-1 is required for ALIS formation, and that the free iron released on heme degradation is necessary and sufficient to induce ALIS. Moreover, ferritin, a key protein in iron metabolism, prevents excessive ALIS formation. Finally, in vivo, hemolysis promotes an increase in ALIS formation in target tissues. Our data unravel a poorly understood aspect of the cellular responses induced by heme that can be explored to better understand the effects of free heme and free iron during hemolytic diseases such as sickle cell disease, dengue fever, malaria, and sepsis.autophagy | p62/SQSTM1 | ALIS | heme | iron

Retention of intracellular Toll-Like Receptors (TLRs) in the endoplasmic reticulum prevents their activation under basal conditions. TLR9 is activated by sensing ligands in specific endosomal/lysosomal compartments. Here, we describe the identification of insulin responsive aminopeptidase (IRAP) endosomes as major cellular compartments for the early steps of TLR9 activation in dendritic cells (DCs). Both TLR9 and its ligand CpG were found as cargo in IRAP endosomes. In the absence of IRAP, CpG and TLR9 trafficking to lysosomes and TLR9 signaling were enhanced in DCs and in mice following bacterial infection. IRAP stabilized CpG-containing endosomes by interacting with the actin nucleation factor FHOD4, slowing down TLR9 trafficking towards lysosomes. Thus, endosome retention of TLR9 via IRAP interaction with the actin cytoskeleton is a mechanism that prevents TLR9hyper-activation in DCs. discriminate between different classes of microbial products and initiate specific signaling cascades. While microbial products with no equivalent in mammalian cells, such as the components of the bacterial wall, are recognized by surface TLRs (1, 2, 4, 5 and 6), pathogen derived nucleic acids are sensed by intracellular TLRs (3,7, 8 and 9). Recognition of nucleic acids by intracellular TLRs has the potential to trigger autoimmune diseases through interaction with self nucleic acids 1 . To avoid inappropriate activation of endosomal TLRs, their trafficking is tightly controlled. Thus, in basal conditions the receptors are located in the endoplasmic reticulum (ER) and translocate to endocytic vesicles only after cell stimulation by TLR ligands. Although all intracellular TLRs reside in the ER 2,3 , the trafficking pathways that move the receptors into the endocytic pathway show considerable variation among intracellular TLRs 4-6 . For example, TLR7 traffics from Golgi stacks directly to endosomes using the clathrin adaptor AP4, whereas the TLR9 is directed to the cell surface and reaches the endosomes via AP2-mediated clathrin-dependent endocytosis 6 .In addition to the transfer into the endocytic pathway, a second step that controls the activation of endosomal TLRs is their partial proteolysis by an array of different proteases, specific for each TLR 5,7-12 .Although less often mentioned, the intracellular trafficking of its ligand also controls the activation of TLR9. TLR9 ligands (CpG) are internalized via clathrin-mediated endocytosis in early endosomes and translocate to late LAMP + compartments 2 . TLR9 activation depends on CpG localization, since the abrogation of CpG translocation to LAMP + compartments by specific inhibitors decreased TLR9 signaling 13,14 . Thus, the intracellular trafficking of both, the ligand and the receptor are essential for the control of TLR9 activation. RESULTS IRAP deletion increases TLR9 responseTo address the role of IRAP in TLRs signaling, wild-type and IRAP-deficient bone marrow derived dendritic cells (BMDCs) were stimulated with specific TLR ligands: polyIC for TLR3, Imiquimod fo...