Supplementary MaterialsSupplementary Document

Supplementary MaterialsSupplementary Document. pattern acknowledgement receptors (PRRs), such as Toll-like receptors (TLRs), which can identify pathogen-associated molecular patterns (PAMPs) and activate downstream signaling cascades to induce inflammation (1). TLR4, the PRR that specifically Haloxon recognizes lipopolysaccharide (LPS) of Gram-negative bacteria, activates nuclear factor kappa light-chain enhancer of activated B cells (NF-B) and mitogen-activated protein kinase (MAPK) through myeloid differentiation factor 88 (MyD88) to induce the expression of proinflammatory cytokines (2, 3). In addition, TLR4 is usually internalized into endosomes upon realizing LPS to induce IRF3-dependent type-I IFN Haloxon production through the adaptor proteins Toll-IL-1 resistance domain-containing adaptor-inducing IFN- (TRIF) and TRAM (TRIF-related adaptor molecule, refs. 4 and 5). However, the molecular mechanism by which innate signaling induces efficient innate responses needs further investigation. Innate Haloxon immune cells, such as macrophages, dendritic cells (DCs), monocytes, and neutrophils, can internalize the invading pathogens by endocytosis. After being internalized, pathogens become targets of a series of vesicular trafficking at organelles ranging from early endosomes to lysosomes, where they are killed by Mst1-Mst2-Rac signaling-induced reactive oxygen species (ROS) and subsequently degraded by hydrolytic enzymes. The antigens derived from those Sele pathogens are then presented on major histocompatibility complex (MHC) molecules, which are subsequently recognized by T cell receptors and activate CD4+ and CD8+ T cells of the adaptive immune system (6). Endocytosis occurs via a variety of mechanisms, specifically clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, and phagocytosis (7, 8). However, the mechanisms linking the endocytosis of invading pathogens and activation of innate signaling remain to be fully explored. Caveolae, a kind of specialized lipid rafts, are bulb-shaped plasma membrane invaginations first explained in the 1950s (9). Since then, caveolae have been reported to be broadly involved in many cell processes, such as endocytosis, transcytosis (a specialized form of endocytosis), lipid homeostasis, and transmission transduction (10, 11). Pathogens that are internalized via caveolae-mediated endocytosis include FimH-expressing (12). Caveolae are also involved in the endocytosis of receptors, such as TLR4 (13, 14), and this is an essential regulatory mechanism for innate immune responses and transmission transduction. The main component proteins of caveolae are caveolins and cavins. Caveolin family consists of three members, namely, caveolin-1 (Cav1), caveolin-2 (Cav2), and caveolin-3 (Cav3). Cav1 and Cav2 are expressed in most cell types (15, 16). Cav3 is usually specifically expressed in muscle mass cells (17). Cav1 in caveolae binds TLR4, endothelial nitric oxide synthase (eNOS), MAPK, cyclooxygenase (COX), and integrin signaling molecules to initiate different signaling pathways (18C21). However, the innate function of Cav1 in bacterial infection and the underlying mechanism are yet to be determined. In this study, we recognized LAPF (lysosome-associated and apoptosis-inducing protein made up of PH and FYVE domains), which was cloned by our laboratory (22, 23), as a Cav1-interacting protein by mass spectrometry. LAPF has been reported to act as an adaptor protein that recruits phosphorylated p53 to lysosomes to trigger lysosomal destabilization during apoptosis (22, 23). We now find that LAPF is usually critically involved in inducing innate immune responses and in enhancing bacterial endocytosis and the bactericide capacity of macrophages by inducing Src-LAPF-Caveolin complex formation. Results Deficiency Impairs Bacterial Endocytosis and Compromises the Bactericidal Ability of Macrophages. To explore the molecular mechanism of caveolae-medicated endocytosis, we first screened for Cav1-interacting proteins by mass spectrometry (MS). LAPF was recognized in the assay as a possible Cav1-interacting protein ((was deleted conditionally and efficiently in macrophages, using the littermate (for 1 h and visualized the internalization of bacteria particles Haloxon using confocal microscopy (Fig. 1and deficiency significantly impaired endocytosis of by macrophages (Fig. 1and (or in comparison with and and deficiency impairs the endocytosis of various pathogen particles by macrophages. Open in a separate windows Fig. 1. and and.