Lipoarabinomannan (LAM) is one of the key virulence factors for Mycobacterium tuberculosis, the etiological agent of tuberculosis. During uptake of mycobacteria, LAM interacts with the cell membrane of the host macrophage and can be detected throughout the cell upon infection. LAM can inhibit phagosomal maturation as well as induce a proinflammatory response in bystander cells. The aim of this study was to investigate how LAM exerts its action on human macrophages. We show that LAM is incorporated into membrane rafts of the macrophage cell membrane via its glycosylphosphatidylinositol anchor and that incorporation of mannosecapped LAM from M. tuberculosis results in reduced phagosomal maturation. This is dependent on successful insertion of the glycosylphosphatidylinositol anchor. LAM does not, however, induce the phagosomal maturation block through activation of p38 mitogen-activated protein kinase, contradicting some previous suggestions.Mycobacterium tuberculosis, the etiological agent of tuberculosis, spreads by aerosol, mainly infecting alveolar macrophages, by which the bacterium is ingested (17). Through inhibition of macrophage functions, the bacterium modulates the host immune response (25). The best-characterized virulence factor of M. tuberculosis, lipoarabinomannan (LAM), is an abundant glycolipid, which is attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and extends through the cell wall of the bacterium (5). During uptake of mycobacteria, LAM interacts with the cell membrane of the host macrophage via specific receptors, including the macrophage mannose receptor and complement receptor 3 (13,14,19), and can then be detected at multiple sites in the cell (27). The host cell exports LAM from the phagosome in an exocytosis-like manner, eliciting responses in bystander cells (1-3). The M. tuberculosis surface harbors mannose-capped LAM (ManLAM), whereas other, less pathogenic, mycobacteria contain LAM either with a phospho-myo-inositol cap (PILAM) or no cap (6). The type of capping and the presence of the GPI anchor is crucial for virulence (13,14). The effects of Man-LAM on the host cell are multiple, but the interference of ManLAM with phagosomal maturation is the best characterized (21) and has been demonstrated in murine and human macrophages (10,12,14).Studies on human lymphocytes showed that LAM localizes to membrane rafts of the lymphocyte membrane, thereby interfering with signaling affecting cytokine production (20).Membrane rafts are cholesterol-and glycosphingolipid-rich domains that act as platforms for cell signaling processes (16). The aim of this study was to investigate whether the effects of LAM are due to incorporation of LAM into membrane rafts of human macrophages. We establish that LAM is incorporated into membrane rafts of the macrophage cell membrane via its GPI anchor, resulting in reduced phagosomal maturation. MATERIALS AND METHODSAntigens and antibodies. ManLAM, cell wall fractions (CWF), and phosphatidylinositol mannosides (PIM) from the virulen...

Lipophosphoglycan (LPG), the major surface glycoconjugate on Leishmania donovani promastigotes, is crucial for the establishment of infection inside macrophages. LPG comprises a polymer of repeating Galβ1,4Manα-PO 4 attached to a lysophosphatidylinositol membrane anchor. LPG is transferred from the parasite to the host macrophage membrane during phagocytosis and induces periphagosomal F-actin accumulation correlating with an inhibition of phagosomal maturation. The biophysical properties of LPG suggest that it may be intercalated into membrane rafts of the host cell membrane. The aim of this study was to investigate if the effects of LPG on phagosomal maturation are mediated via action on membrane rafts. We show that LPG accumulates in rafts during phagocytosis of L. donovani and that disruption of membrane rafts abolished the effects of LPG on periphagosomal F-actin and phagosomal maturation, indicating that LPG requires intact membrane rafts to manipulate host cell functions. We conclude that LPG associates with membrane rafts in the host cell and exert its actions on host-cell actin and phagosomal maturation through subversion of raft function.2 3

The mechanism of how patatin-like phospholipase domain-containing protein 3 (PNPLA3) variant M148 is associated with increased risk of development of hepatic steatosis is still debated. Here, we propose a novel role of PNPLA3 as a key player during autophagosome formation in the process of lipophagy. A human hepatocyte cell line, HepG2 cells, expressing recombinant I148 or 148M, was used to study lipophagy under energy deprived conditions, and lipid droplet morphology was investigated using florescence microscopy, image analysis and biochemical assays. Autophagic flux was studied using the golden-standard of LC3-II turnover in combination with the well characterized GFP-RFP-LC3 vector. To discriminate between, perturbed autophagic initiation and lysosome functionality, lysosomes were characterized by Lysotracker staining and LAMP1 protein levels as well as activity and activation of cathepsin B. For validation, human liver biopsies genotyped for I148 and 148M were analyzed for the presence of LC3-II and PNPLA3 on lipid droplets. We show that the M148-PNPLA3 variant is associated with lipid droplets that are resistant to starvation-mediated degradation. M148 expressing hepatocytes reveal decreased autophagic flux and reduced lipophagy. Both I148-PNPLA3 and M148-PNPLA3 colocalize and interact with LC3-II, but the M148-PNPLA3 variant has lower ability to bind LC3-II. Together, our data indicate that PNPLA3 might play an essential role in lipophagy in hepatocytes and furthermore that the M148-PNPLA3 variant appears to display a loss in this activity, leading to decreased lipophagy.

No abstract

We demonstrated beneficial effects of β-glucan on intestinal barrier function and increased β-glucan-passage through FAE model. Our results provide important and novel knowledge on possible applications of β-glucan in health disorders and diseases characterized by intestinal barrier dysfunction.