Background & Aims Primary sclerosing cholangitis (PSC) is a chronic, immune-mediated cholangiopathy characterized by persistent inflammation and fibrotic remodelling of the biliary tree. We combined spatial proteomics of PSC-affected large bile ducts with a co-culture system of biliary epithelial cells (BECs) and activated peripheral blood mononuclear cells (aPBMCs) to dissect the molecular crosstalk at the epithelial-immune interface. Methods Spatial proteomic analysis was performed on six PSC-affected bile ducts and five healthy controls by GeoMx® DSP. Single-cell RNA-sequencing analysis from publicly available datasets of human control (n = 22) and PSC-affected (n = 8) livers was performed. We developed an in vitro inflammatory model of PSC by transwell co-culture of BECs (i.e. H69 and primary human biliary tree stem cells) and primary human PBMCs (n = 6). Co-cultures were analysed by transcriptomics on nCounter®, by immunoassays and by culture with/without blocking agents. Results Peribiliary glands showed higher PD-L1 expression in PSC (score = 1.2 ± 0.4) compared to controls (0.3 ± 0.5; p <0.05), which was associated with apoptosis in neighbouring immune cells on spatial proteomics. In vitro, aPBMCs induced the upregulation of 68 immunoregulatory genes in BECs; PD-L1 and IL-6 upregulation was confirmed at the protein level. BECs induced increased apoptosis in T (CD4+/CD8+) and NK cells. In co-culture, the exposure of BECs to the IL-6/sIL-6R complex induced significant PD-L1 upregulation (12.7 ± 1.3; p <0.001), which was inhibited by IL-6 receptor blockade (2.6 ± 1.2; p <0.001) or JAK1/2 inhibition. Conclusions These findings define a critical immunosuppressive axis in PSC driven by BEC-derived IL-6 and PD-L1. Mechanistically, IL-6 acts as an upstream regulator of PD-L1 via JAK/STAT3 and NF-κB signalling, predominantly through IL-6 trans-signalling. This study provides a rationale for targeting epithelial-immune cell interactions as a therapeutic strategy in PSC. Impact and implications The pathogenic basis of primary sclerosing cholangitis (PSC) is poorly understood, hampering the development of specific therapeutic approaches. We identified IL-6 as a key upstream regulator of PD-L1 via the JAK/STAT3 and NF-κB pathways in biliary epithelial cells, highlighting its central role in regulating epithelial–immune crosstalk during biliary injury. The identification of the PD-L1 pathway in epithelial–immune cell interactions in PSC may improve understanding of the disease, as well as inform the development of therapeutic strategies.

Defects in lysosomal cholesterol handling provoke fatal disorders presenting neurovisceral symptoms with variable onset and life spans. A prime example is Niemann-Pick type C disease (NPCD), where cholesterol export from the endosomal-lysosomal system is impaired due to variants of either NPC intracellular cholesterol transporter 1 (NPC1) or NPC intracellular cholesterol transporter 2 (NPC2). Therapeutic options for NPCD are limited to palliative care and disease-modifying drugs, and there is a need for new treatments. Here, we explored bromodomain and extra-terminal domain (BET) proteins as new drug targets for NPCD using patient-derived skin fibroblasts. Treatment with JQ1, a prototype BET protein inhibitor, raised the level of NPC1 protein, diminished lysosomal expansion and cholesterol accumulation, and induced extracellular release of lysosomal components in a dose-, time-, and patient-dependent manner. Lastly, JQ1 enhanced and reduced cholesterol accumulation induced by pharmacologic inhibition of NPC1 and of histone deacetylase (HDAC) activity, respectively. Taken together, bromodomain proteins should be further explored as therapeutic drug targets for lysosomal diseases like NPCD, and as new components regulating lysosomal function and cholesterol metabolism.

Background and Aims:Cholangiocarcinoma (CCA) displays remarkable anatomical and histological heterogeneity. Besides diagnosis confirmation, histology currently does not have a major role in the management of CCA. We aimed to study the clinical relevance of histological heterogeneity of CCA and putative tissue biomarkers by creating a multicentric digitalized European CCA Histology Registry.Approach and Results:Nine referral centers, participating in the International Cholangiocarcinoma Clinical Registry, shared samples and data from 293 patients. Histological and immunohistochemistry stains (n=10) were performed. Computed tomography (CT) scans (n=112 cases) were analyzed by morphological and radiomics techniques. A selection of cases (n=18) was processed for spatial transcriptomics analysis. No significant differences in 5-year overall survival (OS) were found in perihilar CCA versus intrahepatic (i) CCA, and in small bile duct (SBD) versus large bile duct (LBD) iCCA. When cases were classified by Periodic acid of Schiff (PAS) positivity (mucin content), PASHIGH LBD iCCA showed a significantly worse 5-year OS compared to PASLOW iCCA. Multivariate Cox regression identified PASHIGH LBD iCCA phenotype as an independent predictor of worse OS. PASHIGH LBD iCCA subtype showed specific molecular characteristics at spatial transcriptomics and immunohistochemistry; CT scans and serology could distinguish PASHIGH LBD iCCA phenotype with excellent accuracy.Conclusions:Our data underline the importance of identifying morphological subclasses with a significant prevalence in CCA as a tool for risk stratification and prognosis. The European CCA Histology Registry represents a valuable platform for integrating digital pathology with clinical, radiological, and molecular information as a framework for digital twin advancement.

Ferroptosis is a form of cellular death involved in the origin, progression, but also regulation of several human diseases. Its regulatory role in the gut-liver-brain axis (GLBA) has not been clarified. Therefore, we sought to summarize the possible correlations between ferroptosis and the GLBA. In this review, we first introduce the phenotype and the main mechanisms of this relatively newly described form of regulated cell death. Then, we analyse the anatomy of the GLBA, describing the connections between the gut and the liver, followed by the anatomical pathways from the gut to the brain and from the liver to the brain. After the morphological aspects, we summarize the main biological modulators of the GLBA, highlighting their physiological and pathological roles. In the end, we discuss in detail the regulatory role of ferroptosis on neuroinflammation and oxidative stress along GLBA, highlighting the key aspects that could be of significant clinical importance as future diagnostic and therapeutic targets.

Much evidence now indicates a direct crosstalk between thyroid hormones (THs) and the immune system. We previously showed that THs behave as anti-inflammatory agents in human leukemic THP-1 monocytes, but a potential neuroprotective effect of THs in microglia have been under-investigated. Microglia, the primary innate immune cells of the brain, play a pivotal role in the regulation of neuroinflammation and nongenomic integrin αvβ3-mediated action of THs appear to be involved. Integrin αvβ3 was highly expressed in activated BV-2 cells, while it was decreased by THs. In addition, THs facilitated wound healing in BV-2 murine microglia cells activated by lipopolysaccharide (LPS), through integrin αvβ3 and reactive oxygen species (ROS), with tetrac (a metabolite of TH) potentiated the inhibition of migration. Nitric oxide (NO) played a role with different mechanisms in the presence of either T3 or T4. Our data showed the capability of TH and analogues to modulate the M1-to-M2 microglial transition and suggest a new spectrum of actions in the central nervous system (CNS).

Background KRAS is the undisputed champion of oncogenes, and despite its prominent role in oncogenesis as mutated gene, KRAS mutation appears infrequent in gliomas. Nevertheless, gliomas are considered KRAS-driven cancers due to its essential role in mouse malignant gliomagenesis. Glioblastoma is the most lethal primary brain tumor, often associated with disturbed RAS signaling. For newly diagnosed GBM, the current standard therapy is alkylating agent chemotherapy combined with radiotherapy. Cisplatin is one of the most effective anticancer drugs and is used as a first-line treatment for a wide spectrum of solid tumors (including medulloblastoma and neuroblastoma) and many studies are currently focused on new delivery modalities of effective cisplatin in glioblastoma. Its mechanism of action is mainly based on DNA damage, inducing the formation of DNA adducts, triggering a series of signal-transduction pathways, leading to cell-cycle arrest, DNA repair and apoptosis. Methods Long-term cultures of human glioblastoma, U87MG and U251MG, were either treated with cis-diamminedichloroplatinum (cisplatin, CDDP) and/or MEK-inhibitor PD98059. Cytotoxic responses were assessed by cell viability (MTT), protein expression (Western Blot), cell cycle (PI staining) and apoptosis (TUNEL) assays. Further, gain-of-function experiments were performed with cells over-expressing mutated hypervariable region (HVR) KRAS G12V plasmids. Results Here, we studied platinum-based chemosensitivity of long-term cultures of human glioblastoma from the perspective of KRAS expression, by using CDDP and MEK-inhibitor. Endogenous high KRAS expression was assessed at transcriptional (qPCR) and translational levels (WB) in a panel of primary and long-term glioblastoma cultures. Firstly, we measured immediate cellular adjustment through direct regulation of protein concentration of K-Ras4B in response to cisplatin treatment. We found increased endogenous protein abundance and involvement of the effector pathway RAF/MEK/ERK mitogen-activated protein kinase (MAPK) cascade. Moreover, as many MEK inhibitors are currently being clinically evaluated for the treatment of high-grade glioma, so we concomitantly tested the effect of the potent and selective non-ATP-competitive MEK1/2 inhibitor (PD98059) on cisplatin-induced chemosensitivity in these cells. Cell-cycle phase distribution was examined using flow cytometry showing a significant cell-cycle arrest in both cultures at different percentage, which is modulated by MEK inhibition. Cisplatin-induced cytotoxicity increased sub-G1 percentage and modulates G2/M checkpoint regulators cyclins D1 and A. Moreover, ectopic expression of a constitutively active KRAS G12V rescued CDDP-induced apoptosis and different HVR point mutations (particularly Ala 185) reverted this phenotype. Conclusion These findings warrant further studies of clinical applications of MEK1/2 inhibitors and KRAS as ‘actionable target’ of cisplatin-based chemotherapy for glioblastoma.

Alpha-synuclein (α-syn) is widely expressed in presynaptic neuron terminals, and its structural alterations play an important role in the pathogenesis of Parkinson's disease (PD). Aggregated α-syn has been found in brain, in the peripheral nerves of the enteric nervous system (ENS) and in the intestinal neuroendocrine cells during synucleinopathies and inflammatory bowel disorders. In the present study, we evaluated the histomorphological features of murine colon with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, a common model of colitis. Thereafter, we investigated the expression of α-syn, Toll-like receptor 4 (TLR4), choline acetyltransferase (ChAT), vasoactive intestinal peptide (VIP), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), and calcitonin-like receptor (CALCR). Finally, we investigated the presence of phosphorylated α-syn (pS129 α-syn) aggregates and their relationship with inflammatory cells. Colon from TNBS mice showed an increase in inflammatory cells infiltrate and significative changes in the architecture of the intestinal mucosa. α-Syn expression was significantly higher in inflamed colon. VIP was increased in both the mucosa and muscularis externa of TNBS mice, while TH, CGRP, and CALCR were significantly reduced in TNBS mice. Amyloid aggregates of pS129 α-syn were detectable in the ENS, as in the macrophages around the glands of the mucosa correlating with the markers of inflammation. This study describes - for the first time - the altered expression of α-syn and the occurrence of amyloid α-syn aggregates in the inflammatory cells under colitis, supporting the critical role of bowel inflammation in synucleinopathies and the involvement of α-syn in IBD.

BACKGROUND:Different studies have shown the key role of endoplasmic reticulum (ER) stress in autoimmune and chronic inflammatory disorders, as well as in neurodegenerative diseases. ER stress leads to the formation of misfolded proteins which affect the secretion of different cell types that are crucial for the intestinal homeostasis.PURPOSE:In this review, we discuss the role of ER stress and its involvement in the development of inflammatory bowel diseases, chronic conditions that can cause severe damage of the gastrointestinal tract, focusing on the alteration of Paneth cells and goblet cells (the principal secretory phenotypes of the intestinal epithelial cells). ER stress is also discussed in the context of neurodegenerative diseases, in which protein misfolding represents the signature mechanism. ER stress in the bowel and consequent accumulation of misfolded proteins might represent a bridge between bowel inflammation and neurodegeneration along the gut-to-brain axis, affecting intestinal epithelial homeostasis and the equilibrium of the commensal microbiota. Targeting intestinal ER stress could foster future studies for designing new biomarkers and new therapeutic approaches for neurodegenerative disorders.

Acute promyelocytic leukemia (APL) is a hematological disease characterized by the expression of the oncogenic fusion protein PML-RARα. The current treatment approach for APL involves differentiation therapy using all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). However, the development of resistance to therapy, occurrence of differentiation syndrome, and relapses necessitate the exploration of new treatment options that induce differentiation of leukemic blasts with low toxicity. In this study, we investigated the cellular and molecular effects of MK-8776, a specific inhibitor of CHK1, in ATRA-resistant APL cells. Treatment of APL cells with MK-8776 resulted in a decrease in PML-RARα levels, increased expression of CD11b, and increased granulocytic activity consistent with differentiation. Interestingly, we showed that the MK-8776-induced differentiating effect resulted synergic with ATO. We found that the reduction of PML-RARα by MK-8776 was dependent on both proteasome and caspases. Specifically, both caspase-1 and caspase-3 were activated by CHK1 inhibition, with caspase-3 acting upstream of caspase-1. Activation of caspase-3 was necessary to activate caspase-1 and promote PML-RARα degradation. Transcriptomic analysis revealed significant modulation of pathways and upstream regulators involved in the inflammatory response and cell cycle control upon MK-8776 treatment. Overall, the ability of MK-8776 to induce PML-RARα degradation and stimulate differentiation of immature APL cancer cells into more mature forms recapitulates the concept of differentiation therapy. Considering the in vivo tolerability of MK-8776, it will be relevant to evaluate its potential clinical benefit in APL patients resistant to standard ATRA/ATO therapy, as well as in patients with other forms of acute leukemias.

Cholangiocytes, the epithelial cells that line the biliary tree, can proliferate under the stimulation of several factors through both autocrine and paracrine pathways. The cocaine-amphetamine-regulated-transcript (CART) peptide has several physiological functions, and it is widely expressed in several organs. CART increases the survival of hippocampal neurons by upregulating brain-derived neurotrophic factor (BDNF), whose expression has been correlated to the proliferation rate of cholangiocytes. In the present study, we aimed to evaluate the expression of CART and its role in modulating cholangiocyte proliferation in healthy and bile duct ligated (BDL) rats in vivo, as well as in cultured normal rat cholangiocytes (NRC) in vitro. Liver samples from both healthy and BDL (1 week) rats, were analyzed by immunohistochemistry and immunofluorescence for CART, CK19, TrkB and p75NTR BDNF receptors. PCNA staining was used to evaluate the proliferation of the cholangiocytes, whereas TUNEL assay was used to evaluate biliary apoptosis. NRC treated or not with CART were used to confirm the role of CART on cholangiocytes proliferation and the secretion of BDNF. Cholangiocytes proliferation, apoptosis, CART and TrkB expression were increased in BDL rats, compared to control rats. We found a higher expression of TrkB and p75NTR, which could be correlated with the proliferation rate of biliary tree during BDL. The in vitro study demonstrated increased BDNF secretion by NRC after treatment with CART compared with control cells. As previously reported, proliferating cholangiocytes acquire a neuroendocrine phenotype, modulated by several factors, including neurotrophins. Accordingly, CART may play a key role in the remodeling of biliary epithelium during cholestasis by modulating the secretion of BDNF.

Several studies have supported the association between maternal immune activation (MIA) caused by exposure to pathogens or inflammation during critical periods of gestation and an increased susceptibility to the development of various psychiatric and neurological disorders, including autism and other neurodevelopmental disorders (NDDs), in the offspring. In the present work, we aimed to provide extensive characterization of the short- and long-term consequences of MIA in the offspring, both at the behavioral and immunological level. To this end, we exposed Wistar rat dams to Lipopolysaccharide and tested the infant, adolescent and adult offspring across several behavioral domains relevant to human psychopathological traits. Furthermore, we also measured plasmatic inflammatory markers both at adolescence and adulthood. Our results support the hypothesis of a deleterious impact of MIA on the neurobehavioral development of the offspring: we found deficits in the communicative, social and cognitive domains, together with stereotypic-like behaviors and an altered inflammatory profile at the systemic level. Although the precise mechanisms underlying the role of neuroinflammatory states in neurodevelopment need to be clarified, this study contributes to a better understanding of the impact of MIA on the risk of developing behavioral deficits and psychiatric illness in the offspring.

Niemann-Pick type C disease (NPCD) is a lysosomal disorder whith patients presenting highly variable onset, neurovisceral symptoms and life spans due to defective lipid homeostasis. At present, therapeutic options are limited to palliative and disease-modifying drugs, and there is a continued need for new approaches. Here, we explored bromodomain and extraterminal (BET) proteins as new drug target for NPCD using patient-derived fibroblasts. Treatment of cells with JQ1, a prototype BET inhibitor, enhanced the level of NPC1 protein and increased its presence in lysosomes, diminished lysosomal expansion and cholesterol accumulation and induced extracellular release of lysosomal components in a time- and dose-dependent manner. The reversal of pathologic changes in an established cell model suggest inhibition of BET protein as new therapeutic approach for NPCD.### Competing Interest StatementMS and VP are listed as inventors on an Italian patent related to the therapeutic use of BET proteins.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase holoenzyme, which adds telomeric DNA repeats on chromosome ends to counteract telomere shortening. In addition, there is evidence of TERT non-canonical functions, among which is an antioxidant role. In order to better investigate this role, we tested the response to X-rays and H2O2 treatment in hTERT-overexpressing human fibroblasts (HF-TERT). We observed in HF-TERT a reduced induction of reactive oxygen species and an increased expression of the proteins involved in the antioxidant defense. Therefore, we also tested a possible role of TERT inside mitochondria. We confirmed TERT mitochondrial localization, which increases after oxidative stress (OS) induced by H2O2 treatment. We next evaluated some mitochondrial markers. The basal mitochondria quantity appeared reduced in HF-TERT compared to normal fibroblasts and an additional reduction was observed after OS; nevertheless, the mitochondrial membrane potential and morphology were better conserved in HF-TERT. Our results suggest a protective function of TERT against OS, also preserving mitochondrial functionality.

Targeting tumor cell metabolism is a new frontier in cancer management. Thus, metabolic pathway inhibitors could be used as anti-estrogen receptor α (ERα) breast cancer (BC) drugs. Here, the interplay among metabolic enzyme(s), the ERα levels and cell proliferation was studied. siRNA-based screen directed against different metabolic proteins in MCF10a, MCF-7 and MCF-7 cells genetically resistant to endocrine therapy (ET) drugs and metabolomic analyses in numerous BC cell lines unveil that the inhibition of GART, a key enzyme in the purine de novo biosynthetic pathway, induces ERα degradation and prevent BC cell proliferation. We report here that a reduced GART expression correlates with a longer relapse-free-survival (RFS) in women with ERα-positive BCs. ERα-expressing luminal A invasive ductal carcinomas (IDCs) are sensitive to GART inhibition and GART expression is increased in receptor-positive IDCs of high grade and stage and plays a role in the development of ET resistance. Accordingly, GART inhibition reduces ERα stability and cell proliferation in IDC luminal A cells where it deregulates 17β-estradiol (E2):ERα signaling to cell proliferation. Moreover, the GART inhibitor lometrexol (LMX) and drugs approved for clinical treatment of primary and metastatic BC (4OH-tamoxifen and the CDK4/CDK6 inhibitors) exert synergic antiproliferative effects in BC cells. In conclusion, GART inhibition by LMX or other inhibitors of the de novo purine biosynthetic pathway could be a novel effective strategy for the treatment of primary and metastatic BCs.

Thyroid hormones, T 3 (triiodothyronine) and T 4 (thyroxine), induce a variety of long-term effects on important physiological functions, ranging from development and growth to metabolism regulation, by interacting with specific nuclear or cytosolic receptors. Extranuclear or nongenomic effects of thyroid hormones are mediated by plasma membrane or cytoplasmic receptors, mainly by αvβ3 integrin, and are independent of protein synthesis. A wide variety of nongenomic effects have now been recognized to be elicited through the binding of thyroid hormones to this receptor, which is mainly involved in angiogenesis, as well as in cell cancer proliferation. Several signal transduction pathways are modulated by thyroid hormone binding to αvβ3 integrin: protein kinase C, protein kinase A, Src, or mitogen-activated kinases. Thyroid hormone-activated nongenomic effects are also involved in the regulation of Na + -dependent transport systems, such as glucose uptake, Na + /K + -ATPase, Na + /H + exchanger, and amino acid transport System A. Of note, the modulation of these transport systems is cell-type and developmental stage-dependent. In particular, dysregulation of Na + /K + -ATPase activity is involved in several pathological situations, from viral infection to cancer. Therefore, this transport system represents a promising pharmacological tool in these pathologies.

Additional file 2. Supplementary Table 1

Previously, we found that telaprevir (Tel), the inhibitor of hepatitis C virus NS3/4A serine protease, reduces estrogen receptor α (ERα) content at the transcriptional level without binding to the receptor, prevents ERα transcriptional activity, and inhibits basal and 17β‐estradiol (E2)‐dependent cell proliferation in different breast cancer (BC) cell lines. Here, we further characterize the Tel action mechanisms on ERα levels and function, identify a possible molecular target of Tel in BC cells, and evaluate Tel as an antiproliferative agent for BC treatment. Tel‐dependent reduction in ERα levels and function depends on a Tel‐dependent decrease in FOXA1 levels and activity. The effect of Tel is transduced by the IGF1‐R/AKT/FOXA1 pathway, with the antiviral compound interacting with IGF1‐R. Tel prevents the proliferation of several BC cell lines, while it does not affect the proliferation of normal nontransformed cell lines, and its antiproliferative effect is correlated with the ratio of FOXA1/IGF1‐R expression. In conclusion, Tel interferes with the IGF1‐R/AKT/FOXA1 pathway and induces cell death in ERα‐expressing BC cells. Thus, we propose that this antiviral could be repurposed for the treatment of ERα‐expressing BC.

Abstract Background Challenges exist in the clinical treatment of luminal estrogen receptor α (ERα)-positive breast cancers (BCs) both to prevent resistance to endocrine therapy (ET) and to treat ET-resistant metastatic BCs (MBC). Therefore, we evaluated if kinases could be new targets for the treatment of luminal primary and MBCs. Methods ~ 170 kinase inhibitors were applied to MCF-7 cells either with adaptative or genetic resistance to ET drugs and both ERα levels and cell proliferation were measured. Robust-Z-score calculation identified AZD7762 (CHK1/CHK2 inhibitor) as a positive hit. Subsequently, Kaplan–Meier analyses of CHK1 and CHK2 impact on ERα-positive BC patients relapse-free-survival (RFS), bioinformatic evaluations of CHK1 and CHK2 expression and activation status as a function of ERα activation status as well as drug sensitivity studies in ERα-positive BC cell lines, validation of the impact of the ATR:CHK1 and ATM:CHK2 pathways on the control of ERα stability and BC cell proliferation via inhibitor- and siRNA-based approaches, identification of the molecular mechanism required for inhibitor-dependent ERα degradation in BC and the impact of CHK1 and CHK2 inhibition on the 17β-estradiol (E2):ERα signaling, synergy proliferation studies between ET-drugs and clinically relevant CHK1 inhibitors in different luminal BC cell lines, were performed. Results A reduced CHK1 expression correlates with a longer RFS in women with ERα-positive BCs. Interestingly, women carrying luminal A BC display an extended RFS when expressing low CHK1 levels. Accordingly, CHK1 and ERα activations are correlated in ERα-positive BC cell lines, and the ATR:CHK1 pathway controls ERα stability and cell proliferation in luminal A BC cells. Mechanistically, the generation of DNA replication stress rather than DNA damage induced by ATR:CHK1 pathway inhibition is a prerequisite for ERα degradation. Furthermore, CHK1 inhibition interferes with E2:ERα signaling to cell proliferation, and drugs approved for clinical treatment of primary and MBC (4OH-tamoxifen and the CDK4/CDK6 inhibitors abemaciclib and palbociclib) exert synergic effects with the CHK1 inhibitors in clinical trials for the treatment of solid tumors (AZD7762, MK8776, prexasertib) in preventing the proliferation of cells modeling primary and MBC. Conclusions CHK1 could be considered as an appealing novel pharmacological target for the treatment of luminal primary and MBCs.

Plasmacytoid dendritic cells (pDCs) are a unique dendritic cell subset specialized in type I interferon production, whose role in Human Immunodeficiency Virus (HIV) infection and pathogenesis is complex and not yet well defined. Considering the crucial role of the accessory protein Nef in HIV pathogenicity, possible alterations in intracellular signalling and extracellular vesicle (EV) release induced by exogenous Nef on uninfected pDCs have been investigated. As an experimental model system, a human plasmacytoid dendritic cell line, GEN2.2, stimulated with a myristoylated recombinant NefSF2 protein was employed. In GEN2.2 cells, Nef treatment induced the tyrosine phosphorylation of STAT-1 and STAT-2 and the production of a set of cytokines, chemokines and growth factors including IP-10, MIP-1β, MCP-1, IL-8, TNF-α and G-CSF. The released factors differed both in type and amount from those released by macrophages treated with the same viral protein. Moreover, Nef treatment slightly reduces the production of small EVs, and the protein was found associated with the small (size < 200 nm) but not the medium/large vesicles (size > 200 nm) collected from GEN2.2 cells. These results add new information on the interactions between this virulence factor and uninfected pDCs, and may provide the basis for further studies on the interactions of Nef protein with primary pDCs.