T. solium is also called “pork tapeworm” that infects both human and pigs. The parasite causes taeniasis in its adult form when lodged to gastrointestinal tract, and cysticercosis when larval form infects muscle or CNS (NCC). The larval infection of the parasite is well established as the major reason for causing acquired epilepsy throughout the endemic regions such as the developing nations like Asian countries, Latin American countries and major regions of Africa (Prasad et al 2008; Garcia et al., 2014). Cellular immune responses in NCC is exceptionally diverse and depends upon the cyst stage, location and the presence of total cyst numbers. The most fascinating part is the viable stage of the cyst where the host remains asymptomatic, which is characterized by the immune evasion and suppression of inflammatory signaling by the parasite derived secretory components (Prasad et al., 2011). In an interesting study from India, viable stage of parasitic infection had been linked with elevated expression of ICAM-1, IL-10 and IL-4, whereas degenerative stage induces more IL-2, IL-1β, TNF-ɑ, IL-6, and IFN-γ suggesting the immunosuppressive nature of intact viable cyst
(Singh et al., 2013). In the same highly endemic population, it was noticed that there was no co-relation between stage, location or number of cysts with the occurrence of seizure (Prasad et al., 2008). Immunosuppression is a broad phenomenon which is achieved by the parasite mediated priming of regulatory cells, skewed inflammatory cell signalling, switching to alternative pathways promoting parasite survival, induction of TH2 responses and/or by inducing cell death pathways or apoptosis of the immune cells (Zakeri et al., 2018; Zakeri A., 2017).
Exosomes are key extra cellular mediators that are secreted by the parasites during infection and contains molecules like lipids, small RNAs, metabolites and proteins which interact with the host immune system and facilitate immunosuppression (Kalluri et al., 2020). Different studies have demonstrated the role of helminths derived secretory vesicles with the immune evasion features which motivated us the investigate the role of T. solium derived exosomes over Mϕ.
In this study, we explored the role of T. solium cyst-derived exosomes over Mϕ. We purified exosomes by ultracentrifugation-based approach and confirmed the presence of exosome using TEM and SEM. Both the tools have been used to characterize the exosomes in previous studies too (Chuo et al., 2018). The TEM and SEM results were further substantiated by nanoparticle tracking analysis (Fig. 1b). Exosomes are lipid bilayer structure, and they tend to fuse with the plasma membrane or are readily internalized by the other cells via endocytic pathway, invariably of their origin like Heligmosomoides polygyrus derived exosomes were found to be efficiently internalized by the macrophage in vitro (Coakley et al., 2017; Gonda et el., 2019). In our study, we also noticed a dose-dependent internalization of CFSE-labelled exosomes by the THP-1 and human Mϕ (Figure S3). This confirmed that T. solium derived exosomes were taken up by Mϕ.
Deciphering the exosome proteins make-up is crucial to understand their effect and way of interaction with host cells (Zhang et al., 2018; Arora et al., 2020). By performing LCMS proteomics analysis, we found these exosomes carry protease like cathepsin L, redox homeostasis like proteins such as superoxide dismutase, thioredoxin 1 and glutathione transferase, ubiquitin transferase protein etc. From their biological process involvement prospective, majority of them are found to be linked with metabolic process such as glutamate metabolism, amino acid metabolism and dicarboxylic acid metabolism, whereas some of them were found to be associated with histone H3 acetylation and chemotaxis related processes (Fig. 2a). At the molecular function level, biomolecules (nucleotide, ATP and anion) binding proteins were detected and those can be associated as the components of RQC complex, TORC2 complex, histone acetyltransferase complex and TORC1 complexes etc. (Fig. 2b-c). These proteomic data revealed novel aspect of T. solium exosomes as they can directly target the metabolic pathways, ribosome associated pathways and redox homeostasis in the host immune cells which are important for survival of parasite in the host.
In response to an external stimulus, PI3K produces phosphatidylinositol(3, 4, 5)triphosphate {(PI(3, 4, 5)P3} that helps to regulate processes such as cell growth, cellular trafficking, defence against pathogens by inducing ROS production, cell survival and can also negatively regulate the apoptotic signaling pathway (Prasad et al., 2011; Song et al., 2016). In this study for the very first time, we had stablished that the exosomes from T. solium impaired PI3K signalling in Mϕ. We observed decrease in AKT activity in a time dependent manner, and it was significantly higher at 24 hours post treatment (Fig. 3c). The direct downstream target of PI3K protein is protein kinase B or AKT which senses the level of PIP3 in the cells and then get phosphorylated, recruited to the plasma membrane via its pleckstrin homology (PH) domain thereby regulate different cellular processes. Our study revealed a very interesting observation, as we did not find any difference in the ratio of phosphor/total AKT but observed a time dependent decline in the total AKT levels. However, surprisingly there was no change in AKT at transcript level (Fig. 3e). Phosphorylation at serine 473 residue is critical for AKT stability (Wei et al., 2018), and we were observing less AKT, so we checked their phosphorylation at serine 473 site. So, we looked for the mode of AKT degradation, whether they are following proteasomal pathway or autophagosomal pathway (Wu et al., 2011, Kim et al., 2021). Although, the major route for protein degradation is proteasomal pathway but we found that exosomes mediated AKT degradation happens via the autophagic flux (Fig. 4c) and the increased lysosomal activity was also found in the stimulated cells. Degradation of protein is accomplished by the selective ubiquitination of target protein, and then they move to proteolytic compartments for degradation (Clague et al, 2010). Increased ubiquitination was also observed in our study as well as AKT selective ubiquitination, and inhibition of final step of autophagy by bafilomycin A1 restored total levels of AKT in the cytosol confirming the role of autophagy in AKT degradation. After establishing AKT degradation we were interested to establish its role in immunological function. Previous studies have linked the ROS generation with AKT pathway, where activation of AKT promotes ROS generation and kills bacteria both in vivo and in vitro (Prasad et al., 2011). ROS pathways are linked to cancer and defence mechanisms against various pathogens (Li et al., 2021). Innate immune cells utilize ROS as the primary defence mechanisms where they generate superoxide radicals in response to microbial invasion and kills the invaders (Spooner et al., 2011). Exosome mediated AKT degradation also resulted in lower ROS production in cells leading to impaired bacterial killing and support our earlier observation (Figure S4a-b).
The role of mTORC1 has been implicated in different processes like, protein synthesis, autophagy inhibition, nutritional status and immune regulation (Liu et al., 2020). Our study also revealed a very novel finding of AKT and mTORC1 degradation by lysosomal pathway under the stimulation of T. solium derived exosomes. Like AKT, exosomes did not affect the phosphorylation of mTORC1 but reduces the total level of mTORC1 in the cells (Fig. 5a). The mTORC1 was previously reported to be selectively degraded via the autophagy flux. Previously, extracellular vesicles from Brugia malayi have been shown to downregulate mTOR signalling in dendritic cells and macrophages (Ricciardi et al., 2021). Further investigation also supported the autophagic flux mediated degradation of mTORC1 in response to exosomes stimulation (Fig. 5c). This was further supported by the ubiquitination of mTORC1 in macrophages and restoration of mTORC1 level in cytosol via bafilomycin A1 treatment (Fig. 5d).
Autophagy gets initiated upon mTORC1 inhibition, which releases ULK1 and then followed by formation of autophagosome (containing cargo) which fuses with lysosomes to form autolysosomes, leading to cargo degradation. LC3I protein is a marker for autophagosome formation, which then lipidated to form LC3II, indicating active process of autophagosome maturation (Yamamoto et al., 2023). On the other side, autophagy process utilises SQSTM1 protein as the selective cargo and its degradation indicates the autolysosome formation. Exosomes actively induces the LC3II formation in Mϕ which is supported by the SQSTM1 degradation and restoration under bafilomycin A1 treatment (Fig. 6e). Autophagy has been linked with reducing the neutral lipid pool by degradation. Both human and THP-1 derived Mϕ showed reduced levels of lipids under exosomes stimulation (Fig. 6c). Similar phenomenon was also observed in PBMC derived macrophages (Fig. 6d).
AKT has been negatively co-related with autophagy and apoptosis in different cells, and it positively promotes cell growth and function. On the other side, autophagy proceeds with increase in apoptotic levels in cells. Previously Echinococcus multilocularis ESP’s have shown to generate tolerogenic phenotype in primary dendritic cells and apoptosis thus considered as a silent mechanism to induce immunosuppression (Nono et al., 2012; Zakeri A, 2017). Similar phenomenon was also induced by T. solium derived exosomes in both THP-1 derived and PBMC derived Mϕ in our study. This observation was also supported by the increase in apoptosis when we consistently increased the exosomes doses (Figure S6). Then to look for the apoptosis associated pathway we analysed caspases. As, caspases are apoptosis related proteins, and their increased expression induce the intracellular signalling cascades followed by final apoptotic death of the cells (Kesavardhana et al., 2020). Elevated expression of cleaved cas-9 and cleaved cas-3 (Fig. 7a, 7d) in treated THP-1 and human Mϕ conformed the caspase dependent apoptosis after exosome treatment.
Cell-to-cell communication via secretory vesicles is extensively studied, where exosomes are profoundly associated with disease pathogenesis, gene silencing, therapeutics and also in immune signalling pathways (Buzas E. I, 2022). Since gaining much of the attention in the last two decades, extracellular vesicles have shown promising contributions in biomedicals application, drug delivery, vaccine, gene therapy and also in the pathogenesis of cancers and autoimmune diseases (Yáñez-Mó et al., 2015). More recently, an interesting study report, during antigen presentation, B-cells release their telomeres along with Rad51 within the extracellular vesicles which are internalized by the T-cells at immunological synapse followed by the integration of telomere at the T-cell’s chromosomal ends forming the long-lived central memory T-cells (Lanna et al., 2022). The parasite derived exosomes had been found to be involved in of dendritic cell activation, Mϕ polarisation towards anti-inflammatory phenotype, etc. (Coakley et al., 2017; Nicolao et al., 2019).
The immune response generated by T. solium cyst is complex and the outcome of immune response has been associated with clinical presentation. An intense inflammatory response along with degenerating cyst has been associated with occurrence of seizure. Though the immune response to cyst varies widely and hence the severity of the disease (Prasad et al 2008). The impaired immune response against T. solium cystic infection had been reported with probable cause of immune suppression by parasite derived proteins, cyst masking with host proteins, or other factors (Prasad et al., 2008; Terrazas et al., 2013; Garcia et al., 2014). For the very first time, our study explored and established the role of exosome-induced alteration in AKT signalling pathway, whereas most of the studies in other helminths have reported the immune-suppressing features of parasite-derived exosomes but mechanistic detail was missing. Here we reported, T. solium viable cyst-derived exosomes specifically target two central molecules AKT and mTORC1, which contribute in a very significant way in immune signalling, to lysosome mediated degradation but not proteasomal degradation. We also showed the induction of autophagy and apoptosis in both THP-1-derived and human Mϕ. Our study has provided a link between impaired Mϕ response during viable cyst infection. Identifying specific molecule/protein or group of molecules responsible for this phenotype may give us new therapeutic abilities.