Lipotoxicity reduces DDX58/Rig-1 expression and activity leading to impaired autophagy and cell death.

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease globally. NAFLD is a consequence of fat accumulation in the liver leading to lipotoxicity. Increasing evidence has demonstrated the critical role of autophagy in NAFLD. This study uncovers the unexpected role of immune surveillance protein DDX58/Rig-1 (DExD/H box helicase 58) in activating macroautophagy/autophagy and protecting from lipotoxicity associated with NAFLD. Here we show for the first time that DDX58 protein is significantly reduced in nonalcoholic steatohepatitis (NASH) mouse model, an aggressive form of NAFLD characterized by inflammation and fibrosis of the liver. In addition to decreased expression of DDX58, we found that DDX58 activity can be attenuated by treatments with palmitic acid (PA), a saturated fatty acid. To investigate whether PA inhibition of DDX58 is harmful to the cell, we characterized DDX58 function in hepatocytes when exposed to high doses of PA in the presence and/or absence of DDX58. We show that siRNA knockdown of DDX58 promotes apoptosis. Importantly, we show that stable overexpression of DDX58 is protective against toxic levels of PA and stimulates autophagy. This study begins to demonstrate the regulation of the autophagy receptor protein SQSTM1/p62 through DDX58. DDX58 expression directly influences SQSTM1 mRNA and protein levels. This work proposes a model in which activating DDX58 increases an autophagic response and this aids in clearing toxic lipid inclusion bodies, which leads to inflammation and apoptosis. Activating a DDX58-induced autophagy response may be a strategy for treating NAFLD.Abbreviations:5'pppdsRNA: 5' triphosphate double-stranded RNA; CDAHFD: choline-deficient, L-amino acid defined high-fat diet; CEBPB: CCAAT/enhancer binding protein (C/EBP), beta; CQ: chloroquine; DDX58/retinoic acid inducible gene 1/Rig-1: DExD/H box helicase 58; h: hours; IFIH1/MDA5: interferon induced with helicase C domain 1; IFNB/IFN-ß: interferon beta 1, fibroblast; KO: knockout; MAVS: mitochondrial antiviral signaling protein; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; PA: palmitic acid; poly:IC: polyinosinic:polycytidylic acid; PRR: pattern recognition receptors; PSR: picrosirus red; RAP: rapamycin; RLR: RIG-I-like receptor; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK-binding kinase 1.

Immunization with merozoite surface protein 2 fused to a Plasmodium-specific carrier protein elicits strain-specific and strain-transcending, opsonizing antibody.

Vaccine trials and cohort studies in Plasmodium falciparum endemic areas indicate that naturally-acquired and vaccine-induced antibodies to merozoite surface protein 2 (MSP2) are associated with resistance to malaria. These data indicate that PfMSP2 has significant potential as a component of a multi-antigen malaria vaccine. To overcome challenges encountered with subunit malaria vaccines, we established that the use of highly immunogenic rPfMSP8 as a carrier protein for leading vaccine candidates rPfMSP119 and rPfs25 facilitated antigen production, minimized antigenic competition and enhanced induction of functional antibodies. We applied this strategy to optimize a rPfMSP2 (3D7)-based subunit vaccine by producing unfused rPfMSP2 or chimeric rPfMSP2/8 in Escherichia coli. rPfMSP2 formed fibrils, which induced splenocyte proliferation in an antigen receptor-independent, TLR2-dependent manner. However, fusion to rPfMSP8 prevented rPfMSP2 amyloid-like fibril formation. Immunization of rabbits elicited high-titer anti-PfMSP2 antibodies that recognized rPfMSP2 of the 3D7 and FC27 alleles, as well as native PfMSP2. Competition assays revealed a difference in the specificity of antibodies induced by the two rPfMSP2-based vaccines, with evidence of epitope masking by rPfMSP2-associated fibrils. Rabbit anti-PfMSP2/8 was superior to rPfMSP2-elicited antibody at opsonizing P. falciparum merozoites for phagocytosis. These data establish rPfMSP8 as an effective carrier for a PfMSP2-based subunit malaria vaccine.