Discordant signaling and autophagy response to fasting in hearts of obese mice: Implications for ischemia tolerance.

Autophagy is regulated by nutrient and energy status and plays an adaptive role during nutrient deprivation and ischemic stress. Metabolic syndrome (MetS) is a hypernutritive state characterized by obesity, dyslipidemia, elevated fasting blood glucose levels, and insulin resistance. It has also been associated with impaired autophagic flux and larger-sized infarcts. We hypothesized that diet-induced obesity (DIO) affects nutrient sensing, explaining the observed cardiac impaired autophagy. We subjected male friend virus B NIH (FVBN) mice to a high-fat diet, which resulted in increased weight gain, fat deposition, hyperglycemia, insulin resistance, and larger infarcts after myocardial ischemia-reperfusion. Autophagic flux was impaired after 4 wk on a high-fat diet. To interrogate nutrient-sensing pathways, DIO mice were subjected to overnight fasting, and hearts were processed for biochemical and proteomic analysis. Obese mice failed to upregulate LC3-II or to clear p62/SQSTM1 after fasting, although mRNA for LC3B and p62/SQSTM1 were appropriately upregulated in both groups, demonstrating an intact transcriptional response to fasting. Energy- and nutrient-sensing signal transduction pathways [AMPK and mammalian target of rapamycin (mTOR)] also responded appropriately to fasting, although mTOR was more profoundly suppressed in obese mice. Proteomic quantitative analysis of the hearts under fed and fasted conditions revealed broad changes in protein networks involved in oxidative phosphorylation, autophagy, oxidative stress, protein homeostasis, and contractile machinery. In many instances, the fasting response was quite discordant between lean and DIO mice. Network analysis implicated the peroxisome proliferator-activated receptor and mTOR regulatory nodes. Hearts of obese mice exhibited impaired autophagy, altered proteome, and discordant response to nutrient deprivation.

Inhibition of the NOD-Like Receptor Protein 3 Inflammasome Is Protective in Juvenile Influenza A Virus Infection.

Influenza A virus (IAV) is a significant cause of life-threatening lower respiratory tract infections in children. Antiviral therapy is the mainstay of treatment, but its effectiveness in this age group has been questioned. In addition, damage inflicted on the lungs by the immune response to the virus may be as important to the development of severe lung injury during IAV infection as the cytotoxic effects of the virus itself. A crucial step in the immune response to IAV is activation of the NOD-like receptor protein 3 (NLRP3) inflammasome and the subsequent secretion of the inflammatory cytokines, interleukin-1ß (IL-1ß), and interleukin-18 (IL-18). The IAV matrix 2 proton channel (M2) has been shown to be an important activator of the NLRP3 inflammasome during IAV infection. We sought to interrupt this ion channel-mediated activation of the NLRP3 inflammasome through inhibition of NLRP3 or the cytokine downstream from its activation, IL-1ß. Using our juvenile mouse model of IAV infection, we show that inhibition of the NLRP3 inflammasome with the small molecule inhibitor, MCC950, beginning 3 days after infection with IAV, improves survival in juvenile mice. Treatment with MCC950 reduces NLRP3 levels in lung homogenates, decreases IL-18 secretion into the alveolar space, and inhibits NLRP3 inflammasome activation in alveolar macrophages. Importantly, inhibition of the NLRP3 inflammasome with MCC950 does not impair viral clearance. In contrast, inhibition of IL-1ß signaling with the IL-1 receptor antagonist, anakinra, is insufficient to protect juvenile mice from IAV. Our findings suggest that targeting the NLRP3 inflammasome in juvenile IAV infection may improve disease outcomes in this age group.

Induction of DISE in ovarian cancer cells in vivo.

The death receptor CD95/Fas can be activated by immune cells to kill cancer cells. shRNAs and siRNAs derived from CD95 or CD95 ligand (CD95L) are highly toxic to most cancer cells. We recently found that these sh/siRNAs kill cancer cells in the absence of the target by targeting the 3'UTRs of critical survival genes through canonical RNAi. We have named this unique form of off-target effect DISE (for death induced by survival gene elimination). DISE preferentially kills transformed cells and cancer stem cells. We demonstrate that DISE induction occurs in cancer cells in vivo after introducing a lentiviral CD95L derived shRNA (shL3) into HeyA8 ovarian cancer cells grown as i.p. xenografts in mice, when compared to a scrambled shRNA. To demonstrate the possibility of therapeutically inducing DISE, we coupled siRNAs to templated lipoprotein nanoparticles (TLP). In vitro, TLPs loaded with a CD95L derived siRNA (siL3) selectively silenced a biosensor comprised of Venus and CD95L ORF and killed ovarian cancer cells. In vivo, two siRNA-TLPs (siL2-TLP and siL3-TLP) reduced tumor growth similarly as observed for cells expressing the shL3 vector. These data suggest that it is possible to kill ovarian cancer cells in vivo via DISE induction using siRNA-TLPs.

Measuring cardiac autophagic flux in vitro and in vivo.

Autophagy is a lysosomal-dependent catabolic pathway that recycles various cytoplasmic-borne components, such as organelles and proteins, through the lysosomes. This process creates energy and biomolecules that are used to maintain homeostasis and to serve as an energy source under conditions of acute stress. Autophagic flux is a measure of efficiency or throughput of the pathway. Here, we describe a method for determining autophagic flux in vitro and in vivo using the autophagosomal/lysosomal fusion inhibitors chloroquine or bafilomycin A1 and then probing for the autophagosomal marker LC3-II via Western Blot.