Fabio Simbari

In mammalian systems RNA can move between cells via vesicles. Here we demonstrate that the gastrointestinal nematode Heligmosomoides polygyrus, which infects mice, secretes vesicles containing microRNAs (miRNAs) and Y RNAs as well as a nematode Argonaute protein. These vesicles are of intestinal origin and are enriched for homologues of mammalian exosome proteins. Administration of the nematode exosomes to mice suppresses Type 2 innate responses and eosinophilia induced by the allergen Alternaria. Microarray analysis of mouse cells incubated with nematode exosomes in vitro identifies Il33r and Dusp1 as suppressed genes, and Dusp1 can be repressed by nematode miRNAs based on a reporter assay. We further identify miRNAs from the filarial nematode Litomosoides sigmodontis in the serum of infected mice, suggesting that miRNA secretion into host tissues is conserved among parasitic nematodes. These results reveal exosomes as another mechanism by which helminths manipulate their hosts and provide a mechanistic framework for RNA transfer between animal species.

Recent studies have demonstrated that many parasites release extracellular vesicles (EVs), yet little is known about the specific interactions of EVs with immune cells or their functions during infection. We show that EVs secreted by the gastrointestinal nematode Heligmosomoides polygyrus are internalized by macrophages and modulate their activation. EV internalization causes downregulation of type 1 and type 2 immune-response-associated molecules (IL-6 and TNF, and Ym1 and RELMα) and inhibits expression of the IL-33 receptor subunit ST2. Co-incubation with EV antibodies abrogated suppression of alternative activation and was associated with increased co-localization of the EVs with lysosomes. Furthermore, mice vaccinated with EV-alum generated protective immunity against larval challenge, highlighting an important role in vivo. In contrast, ST2-deficient mice are highly susceptible to infection, and they are unable to clear parasites following EV vaccination. Hence, macrophage activation and the IL-33 pathway are targeted by H. polygyrus EVs, while neutralization of EV function facilitates parasite expulsion.

Extracellular vesicles (EVs) mediate communication between cells and organisms across all 3 kingdoms of life. Several reports have demonstrated that EVs can transfer molecules between phylogenetically diverse species and can be used by parasites to alter the properties of the host environment. Whilst the concept of vesicle secretion and uptake is broad reaching, the molecular composition of these complexes is expected to be diverse based on the physiology and environmental niche of different organisms. Exosomes are one class of EVs originally defined based on their endocytic origin, as these derive from multivesicular bodies that then fuse with the plasma membrane releasing them into the extracellular environment. The term exosome has also been used to describe any small EVs recovered by high-speed ultracentrifugation, irrespective of origin since this is not always well characterized. Here, we use comparative global lipidomic analysis to examine the composition of EVs, which we term exosomes, that are secreted by the gastrointestinal nematode, Heligmosomoides polygyrus, in relation to exosomes secreted by cells of its murine host. Ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) analysis reveals a 9- to 62-fold enrichment of plasmalogens, as well as other classes of ether glycerophospholipids, along with a relative lack of cholesterol and sphingomyelin (SM) in the nematode exosomes compared with those secreted by murine cells. Biophysical analyses of the membrane dynamics of these exosomes demonstrate increased rigidity in those from the nematode, and parallel studies with synthetic vesicles support a role of plasmalogens in stabilizing the membrane structure. These results suggest that nematodes can maintain exosome membrane structure and integrity through increased plasmalogens, compensating for diminished levels of other lipids, including cholesterol and SM. This work also illuminates the prevalence of plasmalogens in some EVs, which has not been widely reported and could have implications for the biochemical or immunomodulatory properties of EVs. Further comparative analyses such as those described here will shed light on diversity in the molecular properties of EVs that enable them to function in cross-species communication.

The acid sphingomyelinase (aSMase) gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase), via differential trafficking of a common protein precursor. However, the regulation of S-SMase and its role in cytokine-induced ceramide formation remain ill defined. To determine the role of S-SMase in cellular sphingolipid metabolism, MCF7 breast carcinoma cells stably transfected with V5-aSMase(WT) were treated with inflammatory cytokines. Interleukin-1β and tumor necrosis factor-α induced a time- and dose-dependent increase in S-SMase secretion and activity, coincident with selective elevations in cellular C(16)-ceramide. To establish a role for S-SMase, we utilized a mutant of aSMase (S508A) that is shown to retain L-SMase activity, but is defective in secretion. MCF7 expressing V5-aSMase(WT) exhibited increased S-SMase and L-SMase activity, as well as elevated cellular levels of specific long-chain and very long-chain ceramide species relative to vector control MCF7. Interestingly, elevated levels of only certain very long-chain ceramides were evident in V5-aSMase(S508A) MCF7. Secretion of the S508A mutant was also defective in response to IL-1β, as was the regulated generation of C(16)-ceramide. Taken together, these data support a crucial role for Ser(508) in the regulation of S-SMase secretion, and they suggest distinct metabolic roles for S-SMase and L-SMase.

Acid sphingomyelinase (A-SMase) is an important enzyme in sphingolipid metabolism and plays key roles in apoptosis, immunity, development, and cancer. In addition, it mediates cytotoxicity of cisplatin and some other chemotherapeutic drugs. The mechanism of A-SMase activation is still undefined. We now demonstrate that, upon CD95 stimulation, A-SMase is activated through translocation from intracellular compartments to the plasma membrane in an exocytic pathway requiring the t-SNARE protein syntaxin 4. Indeed, down-regulation of syntaxin 4 inhibits A-SMase translocation and activation induced by CD95 stimulation. This leads to inhibition of the CD95-triggered signaling events, including caspase 3 and 9 activation and apoptosis, activation of the survival pathway involving the protein kinase Akt, and important changes in cell cycle and proliferation. The molecular interaction between A-SMase and syntaxin 4 was not known and clarifies the mechanism of A-SMase activation. The novel actions of syntaxin 4 in sphingolipid metabolism and exocytosis we describe here define signaling mechanisms of broad relevance in cell pathophysiology.