Myriam Aouadi

Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation

Gene silencing by double-stranded RNA, denoted RNA interference, represents a new paradigm for rational drug design. However, the transformative therapeutic potential of short interfering RNA (siRNA) has been stymied by a key obstacle—safe delivery to specified target cells in vivo. Macrophages are particularly attractive targets for RNA interference therapy because they promote pathogenic inflammatory responses in diseases such as rheumatoid arthritis, atherosclerosis, inflammatory bowel disease and diabetes. Here we report the engineering of β1,3-d-glucan-encapsulated siRNA particles (GeRPs) as efficient oral delivery vehicles that potently silence genes in mouse macrophages in vitro and in vivo. Oral gavage of mice with GeRPs containing as little as 20 μg kg-1 siRNA directed against tumour necrosis factor α (Tnf-α) depleted its messenger RNA in macrophages recovered from the peritoneum, spleen, liver and lung, and lowered serum Tnf-α levels. Screening with GeRPs for inflammation genes revealed that the mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) is a previously unknown mediator of cytokine expression. Importantly, silencing Map4k4 in macrophages in vivo protected mice from lipopolysaccharide-induced lethality by inhibiting Tnf-α and interleukin-1β production. This technology defines a new strategy for oral delivery of siRNA to attenuate inflammatory responses in human disease.

Fat-specific Protein 27, a Novel Lipid Droplet Protein That Enhances Triglyceride Storage

Fat-specific protein (FSP)27/Cidec is most highly expressed in white and brown adipose tissues and increases in abundance by over 50-fold during adipogenesis. However, its function in adipocytes has remained elusive since its discovery over 15 years ago. Here we demonstrate that FSP27/Cidec localizes to lipid droplets in cultured adipocytes and functions to promote lipid accumulation. Ectopically expressed FSP27-GFP surrounds lipid droplets in 3T3-L1 adipocytes and colocalizes with the known lipid droplet protein perilipin. Immunostaining of endogenous FSP27 in 3T3-L1 adipocytes also confirmed its presence on lipid droplets. FSP27-GFP expression also markedly increases lipid droplet size and enhances accumulation of total neutral lipids in 3T3-L1 preadipocytes as well as other cell types such as COS cells. Conversely, RNA interference-based FSP27/Cidec depletion in mature adipocytes significantly stimulates lipolysis and reduces the size of lipid droplets. These data reveal FSP27/Cidec as a novel adipocyte lipid droplet protein that negatively regulates lipolysis and promotes triglyceride accumulation.

Activation of the Nlrp3 inflammasome in infiltrating macrophages by endocannabinoids mediates beta cell loss in type 2 diabetes

Type 2 diabetes mellitus (T2DM) progresses from compensated insulin resistance to beta cell failure resulting in uncompensated hyperglycemia, a process replicated in the Zucker diabetic fatty (ZDF) rat. The Nlrp3 inflammasome has been implicated in obesity-induced insulin resistance and beta cell failure. Endocannabinoids contribute to insulin resistance through activation of peripheral CB1 receptors (CB1Rs) and also promote beta cell failure. Here we show that beta cell failure in adult ZDF rats is not associated with CB1R signaling in beta cells, but rather in M1 macrophages infiltrating into pancreatic islets, and that this leads to activation of the Nlrp3-ASC inflammasome in the macrophages. These effects are replicated in vitro by incubating wild-type human or rodent macrophages, but not macrophages from CB1R-deficient (Cnr1−/−) or Nlrp3−/− mice, with the endocannabinoid anandamide. Peripheral CB1R blockade, in vivo depletion of macrophages or macrophage-specific knockdown of CB1R reverses or prevents these changes and restores normoglycemia and glucose-induced insulin secretion. These findings implicate endocannabinoids and inflammasome activation in beta cell failure and identify macrophage-expressed CB1R as a therapeutic target in T2DM.

Similarity of mouse perivascular and brown adipose tissues and their resistance to diet-induced inflammation

Thoracic perivascular adipose tissue (PVAT) is a unique adipose depot that likely influences vascular function and susceptibility to pathogenesis in obesity and the metabolic syndrome. Surprisingly, PVAT has been reported to share characteristics of both brown and white adipose, but a detailed direct comparison to interscapular brown adipose tissue (BAT) has not been performed. Here we show by full genome DNA microarray analysis that global gene expression profiles of PVAT are virtually identical to BAT, with equally high expression of Ucp-1, Cidea, and other genes known to be uniquely or very highly expressed in BAT. PVAT and BAT also displayed nearly identical phenotypes upon immunohistochemical analysis, and electron microscopy confirmed that PVAT contained multilocular lipid droplets and abundant mitochondria. Compared with white adipose tissue (WAT), PVAT and BAT from C57BL6/J mice fed a high-fat diet for 13 wk had markedly lower expression of immune cell-enriched mRNAs, suggesting resistance to obesity-induced inflammation. Indeed, staining of BAT and PVAT for macrophage markers (F4/80 and CD68) in obese mice showed virtually no macrophage infiltration, and FACS analysis of BAT confirmed the presence of very few CD11b(+)/CD11c(+) macrophages in BAT (1.0%) compared with WAT (31%). In summary, murine PVAT from the thoracic aorta is virtually identical to interscapular BAT, is resistant to diet-induced macrophage infiltration, and thus may play an important role in protecting the vascular bed from inflammatory stress.

Coronin 2A mediates actin-dependent de-repression of inflammatory response genes

Toll-like receptors (TLRs) function as initiators of inflammation through their ability to sense pathogen-associated molecular patterns and products of tissue damage. Transcriptional activation of many TLR-responsive genes requires an initial de-repression step in which nuclear receptor co-repressor (NCoR) complexes are actively removed from the promoters of target genes to relieve basal repression. Ligand-dependent SUMOylation of liver X receptors (LXRs) has been found to suppress TLR4-induced transcription potently by preventing the NCoR clearance step, but the underlying mechanisms remain enigmatic. Here we provide evidence that coronin 2A (CORO2A), a component of the NCoR complex of previously unknown function, mediates TLR-induced NCoR turnover by a mechanism involving interaction with oligomeric nuclear actin. SUMOylated LXRs block NCoR turnover by binding to a conserved SUMO2/SUMO3-interaction motif in CORO2A and preventing actin recruitment. Intriguingly, the LXR transrepression pathway can itself be inactivated by inflammatory signals that induce calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ)-dependent phosphorylation of LXRs, leading to their deSUMOylation by the SUMO protease SENP3 and release from CORO2A. These findings uncover a CORO2A–actin-dependent mechanism for the de-repression of inflammatory response genes that can be differentially regulated by phosphorylation and by nuclear receptor signalling pathways that control immunity and homeostasis.

Insulin signalling mechanisms for triacylglycerol storage.

Insulin signalling is uniquely required for storing energy as fat in humans. While de novo synthesis of fatty acids and triacylglycerol occurs mostly in liver, adipose tissue is the primary site for triacylglycerol storage. Insulin signalling mechanisms in adipose tissue that stimulate hydrolysis of circulating triacylglycerol, uptake of the released fatty acids and their conversion to triacylglycerol are poorly understood. New findings include (1) activation of DNA-dependent protein kinase to stimulate upstream stimulatory factor (USF)1/USF2 heterodimers, enhancing the lipogenic transcription factor sterol regulatory element binding protein 1c (SREBP1c); (2) stimulation of fatty acid synthase through AMP kinase modulation; (3) mobilisation of lipid droplet proteins to promote retention of triacylglycerol; and (4) upregulation of a novel carbohydrate response element binding protein β isoform that potently stimulates transcription of lipogenic enzymes. Additionally, insulin signalling through mammalian target of rapamycin to activate transcription and processing of SREBP1c described in liver may apply to adipose tissue. Paradoxically, insulin resistance in obesity and type 2 diabetes is associated with increased triacylglycerol synthesis in liver, while it is decreased in adipose tissue. This and other mysteries about insulin signalling and insulin resistance in adipose tissue make this topic especially fertile for future research.

Gene silencing in adipose tissue macrophages regulates whole-body metabolism in obese mice

Adipose tissue (AT) inflammation and infiltration by macrophages is associated with insulin resistance and type 2 diabetes in obese humans, offering a potential target for therapeutics. However, whether AT macrophages (ATMs) directly contribute to systemic glucose intolerance has not been determined. The reason is the lack of methods to ablate inflammatory genes expressed in macrophages specifically localized within AT depots, leaving macrophages in other tissues unaffected. Here we report that i.p. administration of siRNA encapsulated by glucan shells in obese mice selectively silences genes in epididymal ATMs, whereas macrophages within lung, spleen, kidney, heart, skeletal muscle, subcutaneous (SubQ) adipose, and liver are not targeted. Such administration of GeRPs to silence the inflammatory cytokines TNF-α or osteopontin in epididymal ATMs of obese mice caused significant improvement in glucose tolerance. These data are consistent with the hypothesis that cytokines produced by ATMs can exacerbate whole-body glucose intolerance.

Glucan particles for selective delivery of siRNA to phagocytic cells in mice.

Phagocytic macrophages and dendritic cells are desirable targets for potential RNAi (RNA interference) therapeutics because they often mediate pathogenic inflammation and autoimmune responses. We recently engineered a complex 5 component glucan-based encapsulation system for siRNA (small interfering RNA) delivery to phagocytes. In experiments designed to simplify this original formulation, we discovered that the amphipathic peptide Endo-Porter forms stable nanocomplexes with siRNA that can mediate potent gene silencing in multiple cell types. In order to restrict such gene silencing to phagocytes, a method was developed to entrap siRNA-Endo-Porter complexes in glucan shells of 2-4 μm diameter in the absence of other components. The resulting glucan particles containing fluorescently labelled siRNA were readily internalized by macrophages, but not other cell types, and released the labelled siRNA into the macrophage cytoplasm. Intraperitoneal administration of such glucan particles containing siRNA-Endo-Porter complexes to mice caused gene silencing specifically in macrophages that internalized the particles. These results from the present study indicate that specific targeting to phagocytes is mediated by the glucan, whereas Endo-Porter peptide serves both to anchor siRNA within glucan particles and to catalyse escape of siRNA from phagosomes. Thus we have developed a simplified siRNA delivery system that effectively and specifically targets phagocytes in culture or in intact mice.

CD40 deficiency in mice exacerbates obesity-induced adipose tissue inflammation, hepatic steatosis, and insulin resistance

The pathophysiology of obesity and type 2 diabetes in rodents and humans is characterized by low-grade inflammation in adipose tissue and liver. The CD40 receptor and its ligand CD40L initiate immune cell signaling promoting inflammation, but conflicting data on CD40L-null mice confound its role in obesity-associated insulin resistance. Here, we demonstrate that CD40 receptor-deficient mice on a high-fat diet display the expected decrease in hepatic cytokine levels but paradoxically exhibit liver steatosis, insulin resistance, and glucose intolerance compared with their age-matched wild-type controls. Hyperinsulinemic-euglycemic clamp studies also demonstrated insulin resistance in glucose utilization by the CD40-null mice compared with wild-type mice. In contrast to liver, adipose tissue in CD40-deficient animals harbors elevated cytokine levels and infiltration of inflammatory cells, particularly macrophages and CD8(+) effector T cells. In addition, ex vivo explants of epididymal adipose tissue from CD40(-/-) mice display elevated basal and isoproterenol-stimulated lipolysis, suggesting a potential increase of lipid efflux from visceral fat to the liver. These findings reveal that 1) CD40-null mice represent an unusual model of hepatic steatosis with reduced hepatic inflammation, and 2) CD40 unexpectedly functions in adipose tissue to attenuate its inflammation in obesity, thereby protecting against hepatic steatosis.

Lipid storage by adipose tissue macrophages regulates systemic glucose tolerance

Proinflammatory pathways in adipose tissue macrophages (ATMs) can impair glucose tolerance in obesity, but ATMs may also be beneficial as repositories for excess lipid that adipocytes are unable to store. To test this hypothesis, we selectively targeted visceral ATMs in obese mice with siRNA against lipoprotein lipase (LPL), leaving macrophages within other organs unaffected. Selective silencing of ATM LPL decreased foam cell formation in visceral adipose tissue of obese mice, consistent with a reduced supply of fatty acids from VLDL hydrolysis. Unexpectedly, silencing LPL also decreased the expression of genes involved in fatty acid uptake (CD36) and esterification in ATMs. This deficit in fatty acid uptake capacity was associated with increased circulating serum free fatty acids. Importantly, ATM LPL silencing also caused a marked increase in circulating fatty acid-binding protein-4, an adipocyte-derived lipid chaperone previously reported to induce liver insulin resistance and glucose intolerance. Consistent with this concept, obese mice with LPL-depleted ATMs exhibited higher hepatic glucose production from pyruvate and glucose intolerance. Silencing CD36 in ATMs also promoted glucose intolerance. Taken together, the data indicate that LPL secreted by ATMs enhances their ability to sequester excess lipid in obese mice, promoting systemic glucose tolerance.