purchase thapsigargin We have previously reported that Nef a

We have previously reported that Nef activates and shuttles activated ADAM17 into extracellular vesicles (EV) (Lee et al., 2013). This occurred through interaction with a multifaceted protein complex termed the Nef-associated kinase complex (NAKC). In more recent results we demonstrated that Nef/ADAM17-containing plasma extracellular vesicles (HIV-pEV) persist in high concentrations despite ART and correlate with low CD4 counts (Lee et al., 2016). Together these findings suggested that Nef has a direct role in HIV immune activation and in AIDS pathogenesis. To substantiate this assumption we were looking for a mechanistic link between the effects of Nef and HIV plasma EV (pEV) and the release of TNF in chronic infection. To this end we analyzed TNF secretion in vitro and in/with human material from ART patients. In addition to current concepts, which describe a plasma membrane (PM)-associated shedding of soluble TNF, we found that the larger pool of proTNF is cleaved intracellularly and secreted through vesicular endosomes. This unusual mechanism required the translocation of ADAM17 into Rab4+ compartments, where the protease converged with its substrate. Analysis of human tissue confirmed these findings and implicated Notch1 as a crucial co-factor in trafficking of ADAM17. We conclude that HIV evolved a powerful TNF-mobilizing mechanism, which persists in ART-patients and may contribute to chronic immune activation.
Material and Methods
Results
Discussion We have previously demonstrated a transfer of Nef and Nef-associated signaling mechanisms to bystander purchase thapsigargin by means of vesicle transfer and trogocytosis (Muratori et al., 2009). We speculated that this transfer of infected cell signaling (TOS) serves to prepare/activate the resting bystander cell for viral infection. The here described vesicular secretion of TNF appears to be part of this function. This type of secretion is mechanistically different from TNF surface shedding as cleaved TNF is kept in the lumen of endosomal vesicles (see Fig. 1d). This excludes a surface to surface interaction, e.g. vesicle to plasma membrane, for TNF receptor stimulation. Hence, this secretion type, which we detailed with electron micrographs in our previous work (Muratori et al., 2009), is different from microvesicle (Cocucci et al., 2009) and Exosome release (Thery et al., 2002) and may be termed endosomal or vesicular secretion. Based on our confocal analysis we suggest that pEV are endocytosed by target cells, possibly in a clathrin-dependent manner. Subsequently they may fuse with endosomes, for example containing inward directed TNF receptor molecules for sustained signaling. Although this is speculation and requires further analysis, we summarized these events in a model, which includes findings from our previous work and the literature (Fig. S7) (Muratori et al., 2009; Gonzalez-Gaitan, 2003; Lee et al., 2013). ADAM17 needed to be internalized into Rab4+ positive EE, in order to converge with its substrate proTNF. This routing was initiated by Nef, most likely through its well described induction of clathrin coated vesicles (CCV) (Mangasarian et al., 1997). The CCV probably transformed into, or fused with, pre-existing Rab4+ and proTNF-containing compartments. These events suggested a spatially separated TNF secretion mechanism, which is not necessarily connected to TNF surface shedding. We speculate that activation stimuli that include internalization signals for ADAM17 (e.g. Nef or PMA) will induce endosomal proTNF cleavage and secretion, whereas signals confined to the plasma membrane will cause primarily TNF shedding from the cell surface. We found that Nef association with signaling molecules (NAKC) was required for endosomal TNF processing, suggesting that Nef-induced endocytosis and Nef signaling serve a common purpose. This function is characterized by two tightly linked steps. First, ADAM17 is activated by a Nef-induced signaling process (Percario et al., 2015; Lee et al., 2013) and, in a second step, shuttled to the proTNF pool in Rab4+ EE. In host cells this is initiated by Nef expression, in bystander cells via Nef EV-ingestion. By which mechanism incoming Nef pEV induce internalization of cell-resident ADAM17 (as in Fig. 3) is not clear. Based on our data we could envision a scenario by which incoming pEV fuse with endosomes containing cellular ADAM17 (Fig. S7).