SIGNAL TRANSDUCTION AND EXTRACELLULAR VESICLES RELEASE INDUCED BY HIV-1 NEF PROTEIN ON PLASMACYTOID DENDRITIC CELLS

Abstract

Plasmacytoid dendritic cells (pDCs) are a unique dendritic cell subset specialized in type I interferon (IFN) production and whose role in HIV-1 infection and pathogenesis is complex and not yet well defined. Although they do not represent the main reservoir of HIV, it is reported that pDCs can be infected by the virus contributing dichotomously to both chronic immune activation and immunosuppression [Aiello et al., 2018]. To date, most of the reported studies have been focused on the analysis of pDC response following HIV infection. However, emerging evidences point out the ability of the viral protein Nef to be transferred through cell-to-cell contact and extracellular vesicles (EVs) [Campbell et al., 2008; Xu et al., 2009; Muratori et al., 2009; Lenassi et al., 2010; Pužar Dominkuš et al., 2017], thus exerting specific effects on both infected and uninfected cells. Moreover, both Nef and anti-Nef antibodies were detected in the serum of HIV-infected individuals [Fujii et al., 1996; Ameisen et al., 1989] supporting the possible in vivo detection of extracellular Nef by uninfected cells. In light of what reported above, in this work the effects induced by the pathogenic accessory protein Nef alone of HIV-1 SF2 strain on HIV-not infected pDCs were characterized. Previous results obtained in our laboratory demonstrated that the recombinant myristoylated Nef protein (myrNefSF2) was rapidly internalized in primary monocyte-derived macrophages (MDMs) and triggered NF-κB, MAPKs (Mitogen-Activated Protein Kinase) and IRF-3 (Interferon Regulatory Factor 3) activation inducing the production and release of a set of cytokines/chemokines including IFN-β [Mangino et al., 2007 and 2011]. The latter, in turn, activated some signal transducers and activators of transcription (STAT) molecules in an autocrine and/or paracrine manner, in particular STAT-1, -2 and -3 [Olivetta et al., 2003; Mangino et al., 2007; Federico et al., 2001; Percario et al., 2003]. Therefore, we started to investigate the possible alterations in intracellular signalling induced by Nef. The preliminary results obtained in primary pDCs revealed that the exogenous treatment with Nef protein up-regulated the expression of mxa, an IFN-inducible gene, whose protein is usually used as surrogate marker for type I and III IFN production. Moreover, confocal images showed that Nef induced the increase and a partial nuclear translocation of IRF-7 that could cause type I IFN production. Since plasmacytoid dendritic cells represent a minor fraction of peripheral blood leukocytes, in order to facilitate biochemical analyses and have a more stable and reproducible system we decided to carry out further analyses using a human plasmacytoid dendritic cell line, GEN2.2. The latter was provided by Dr. Laurence Chaperot through a Material Transfer Agreement with the CNCM (Collection Nationale de Cultures de Microorganismes), Pasteur Institute of Paris. Western blot analyses performed on GEN2.2 revealed that Nef induced the tyrosine phosphorylation of both STAT1 and STAT2 proteins starting from 3 hours. Notably, Nef substantially influenced also the gene expression program via STAT activation, as indicated by the late induction of IRF-1, STAT1 and ISG15. In contrast, the treatment with the Nef mutant 4EA, mutated in the acidic domain of the protein, was not able to induce the phosphorylation of STAT1 and STAT2 proteins and did not even modulate the gene expression since neither IRF-1, STAT1 nor ISG15 were increased. These results highlight the importance of the acidic domain in the signalling pathway induced by Nef and add relevance to the previous findings obtained in primary macrophages [Mangino et al., 2007 and 2011]. Concerning the expression of co-stimulatory molecules such as CD40, CD80 and CD86, which usually accompany the activation or maturation of plasmacytoid dendritic cells making them efficient antigen presenting cells (APCs), it was observed that Nef protein did not alter them thus suggesting that Nef could act on pDCs by favouring the acquisition of an interferon producing cells (IPC) phenotype rather than an APC one. The secretome analysis performed on the supernatants after Nef treatment revealed that the protein induced in GEN2.2 the production of regulatory cytokines (IL-2), growth factors (FGF basic and G CSF), and chemotactic and/or pro-inflammatory mediators (MCP-1, IL-8, IP-10, MIP-1α, MIP-1β, IFN-γ and TNF-α). Other mediators, such as IL-4, IL-5, IL-17, Eotaxin and RANTES resulted to be only weakly secreted in response to Nef treatment. Since macrophages are widely recognized as one of the main reservoir of HIV infection, we compared the pattern of cytokines/chemokines induced by Nef in GEN2.2 with that of THP-1, a monocytic cell line that was differentiated adding PMA in order to acquire a macrophage-like phenotype. Notably, unlike GEN2.2, in THP-1/PMA Nef did not affect or only weakly the expression of IL-8, G-CSF and MCP-1, whereas promoted the secretion of some cytokines weakly induced or not produced in GEN2.2 such as PDGF, IL-1β, IL-5, IL-15, IL-17, RANTES and VEGF. Other mediators such as IL-1ra, IL-4, IL-12 (p70), IFN-γ and Eotaxin were only weakly modulated in THP-1/PMA. Soluble factors such as FGF basic, IL-2, IP-10 and TNF-α were secreted by both cell types, although at different extent. Indeed, TNF-α was much more induced in THP-1/PMA. In conclusion, these results highlight the ability of Nef protein to induce the release of a different pattern of cytokines/chemokines according to the cell type probably contributing to fuel in different ways the intense “cytokine storm” that characterizes HIV infection [Wang et al., 2017]. Furthermore, since during HIV infection pDCs are exposed to the local microenvironment influenced by the immunostimulatory molecules secreted by infected cells into the extracellular space, we verified the response of GEN2.2 to the cytokine/chemokine milieu released by GEN2.2 in response to Nef stimulus. It was observed that STAT1 tyrosine phosphorylation occurred more rapidly (already after 30 minutes) than following Nef treatment (3 hours) showing that pDCs are promptly responsive to the surrounding extracellular milieu. Interestingly, supernatants depleted of extracellular vesicles (EVs) maintained the capacity to early activate STAT1 already after 30 minutes indicating that the cytokines/chemokines responsible for this phenomenon should be mostly secreted in free form and not associated with EVs. Subsequently, considering the emerging importance of the EVs for their role in the intercellular communication in both physiological and pathological conditions, including HIV infection [Dias et al., 2018], we characterized and quantified the EVs (exosomes and microvesicles) released by GEN2.2 in response to Nef treatment. To fulfil our purpose, we used the commercially available Bodipy FL C16 fatty acid to label the cells. The fluorescent EVs released from the cells were examined and quantified as reported by Sargiacomo and colleagues [Coscia et al., 2016]. Interestingly, unlike what reported in literature regarding other cell types, we found that Nef did not increase the production of exosomes in GEN2.2, but it induced a 40% reduction. Moreover, Nef was preferentially incorporated into the exosomal pellet after cell treatment, but not in microvesicles, suggesting the presence of a specific mechanism that would address the protein to be released into exosomes. In addition, we observed an increased expression of the exosomal markers CD81, Tsg101, and Flotillin 1 in exosomes secreted from GEN2.2 treated with Nef possibly reflecting the diversity of the vesicles released in response to Nef treatment compared to those secreted by untreated cells. Finally, considered the particular attitude of pDCs to secrete IFNs and their continuous exposure to these types of cytokines during HIV infection, we analysed how they could influence the number of EVs secreted by pDCs. In this regard, we observed that neither type I, II or III IFN alter significantly the exosome or microvesicle release. However, they seem to affect from a qualitative point of view the type of vesicles released because in response to all IFN types a down-modulated expression of the exosomal markers CD81, Tsg101, and Flotillin-1 was observed. Altogether, the results of this work shed new light on the effects exerted by Nef protein alone on uninfected pDCs by contributing to provide a more comprehensive picture for a thorough understanding of pDCs roles in HIV infection that may help to define pDCs functions and develop therapeutic strategies.