Leptin is an adipocyte-derived hormone/cytokine that links nutrition, metabolism, and immune homeostasis. Leptin is capable of modulating several immune responses. However, the effect of leptin on dendritic cells (DCs) has not yet been recognized. Because DCs are instrumental in the development of immune responses, in this study, we evaluated the impact of leptin on DC activation. We demonstrated the presence of leptin receptor in human immature and mature DCs both at mRNA and protein level and its capacity to transduce leptin signaling leading to STAT-3 phosphorylation. We found no consistent modulation of DC surface molecules known to be critical for their APC function in response to leptin. In contrast, we found that leptin induces rearrangement of actin microfilaments, leading to uropod and ruffle formation. At a functional level, leptin up-regulates the IL-1β, IL-6, IL-12, TNF-α, and MIP-1α production. Coincident with this, leptin-treated DCs stimulate stronger heterologous T cell responses. Furthermore, we found that leptin down-regulates IL-10 production by DCs and drives naive T cell polarization toward Th1 phenotype. Finally, we found that leptin partly protects DCs from spontaneous and UVB-induced apoptosis. Consistent with the antiapoptotic effect of leptin, we observed the activation of NF-κB and a parallel up-regulation of bcl-2 and bcl-xL gene expression. These results provide new insights on the immunoregulatory function of leptin demonstrating its ability to improve DC functions and to promote DC survival. This is of relevance considering a potential application of leptin in immunotherapeutic approaches and its possible use as adjuvant in vaccination protocols.

The accessory HIV-1 Nef protein plays a key role in AIDS pathogenesis. We recently demonstrated that exogenous Nef triggers phenotypic and functional differentiation of immature dendritic cells (DCs). Here we investigated whether the Nef-induced DC differentiation occurs with morphological remodeling and have focused on the interference of Nef in the signaling pathways that regulates DC maturation. We found that exogenous Nef enters immature DCs, promoting their functional and morphological differentiation. Specifically, Nef promotes interleukin (IL) -12 release, which closely fits with nuclear factor (NF) -kappaB activation. Nef induces rearrangement of actin microfilaments, leading to uropod and ruffle formation. Moreover, Nef increases the capacity of DCs to form clusters with allogeneic CD4+ T cells, improving immunological synapse formation. Searching for molecules involved in Nef-triggered signaling pathways driving the DC maturation, we found that Nef targets Vav and promotes its tyrosine phosphorylation, associated with its nucleus-to-cytoplasm redistribution. This has a direct effect on Vav guanine nucleotide exchange factor activity for the small GTPase Rac1. We hypothesize that targeting Vav, Nef modulates both early signaling events (such as cytoskeletal rearrangement) and delayed responses (such as transcriptional regulation), promoting DC differentiation. Our results highlight how Nef may enhance T lymphocyte activation, thus fostering virus dissemination, manipulating the DC arm of the immune response.

TLRs are a family of molecules that function as sensors for the detection of pathogens. TLR-9, expressed on B cells and pDCs, recognizes CpG motifs of unmethylated bacterial DNA and plays a role in the development of autoimmunity. The present study was designed to investigate the effects of IFN-alpha in combination with CpG ODN on the activation of CD27(-) naïve B cells and on Ig production. We provide evidence that CpG ODN not only induces a total and T-dependent, specific IgM response by naïve B cells but also their phenotypic differentiation in plasma cells, as demonstrated by the up-regulation of CD38 expression. We found that TLR-9 stimulation with CpG ODN induces IL-1beta, TNF-alpha, IL-10, and IL-6 production. Interestingly, we also found that CpG ODN induces naïve B cell maturation into memory cells, as demonstrated by the induction of CD27, AID mRNA expression, and IgG production. More importantly, our results demonstrate that IFN-alpha amplifies the inductive effect of CpG ODN on naïve B activation and on Ig production through a mechanism involving TLR-9/MyD88-dependent signaling. Moreover, we found that IFN-alpha enhances the frequency of CpG ODN-induced memory B cells. Our results may contribute to clarify the events promoting IFN-alpha-induced amplification of naïve B cell activation via TLR-9 for a better understanding of the pathogenesis of autoimmune disorders and may guide treatments targeting this pathway within B cells.

The aim of the present work was to evaluate the contribution of the different reactive oxidizing species to systemic oxidative stress in the whole blood of patients with Kawasaki disease (KD). This is a rare generalized systemic vasculitis typical of the early childhood characterized by inflammation and endothelial dysfunction with a high risk for cardiovascular fatal events. We found that, compared to age-matched healthy donors, blood from KD patients showed increased production of oxygen- and nitrogen-derived species as detected by electron paramagnetic resonance (EPR) spin probing with the cyclic hydroxylamine 1-hydroxy-3-carboxy-pyrrolidine. The (•)NO pathway involvement was also confirmed by the decreased concentrations of the endogenous (•)NO synthase inhibitor asymmetric dimethyl-arginine and the increased amounts of 3-nitrotyrosine in plasma. Further, increased plasma yields of the proinflammatory enzyme myeloperoxidase were also observed. The appearance of circulating red blood cell alterations typically associated with oxidative imbalance and premature aging (e.g., decrease of total thiol content, glycophorin A, and CD47 expression, as well as increase of phosphatidylserine externalization) has also been detected. Collectively, our observations lead to hypothesize that the simultaneous oxidative and nitrative stress occurrence in the blood of KD patients may play a pathogenetic role in the cardiovascular complications often associated with this rare disease.

a b s t r a c tLeptin is an adipocyte-derived hormone/cytokine that modulates immune responses. It induces functional and morphological changes in human dendritic cells (DCs), licensing them towards Th1 priming and promoting DC survival. Here we found that leptin protects DCs from spontaneous, UVB and H 2 O 2 -induced apoptosis, by triggering the activation of nuclear factor-kappa B (NF-kB) and a parallel up-regulation of bcl-2 and bcl-XL gene expression and Akt activation. We found that leptin activates the PI3K-Akt signaling pathway as demonstrated by the suppression of the effect of leptin on DC survival by wortmannin and API-2, which suppress the leptin-induced activation of Akt, NFkB, bcl-2, bcl-XL and protection from apoptosis. These results provide insights on the immunoregulatory function of leptin, supporting a potential application in immunotherapeutic approaches.

Leptin is an adipocyte-derived hormone/cytokine that links nutrition, metabolism, and immune homeostasis and is endowed to modulate several immune responses. We previously demonstrated that both immature and mature human dendritic cells (DCs) express a functional leptin receptor, and we found that leptin activates DCs, licenses them for Th1 priming, and promotes DC survival. Moreover, we found that leptin induces rearrangement of actin microfilaments, leading to uropod and ruffle formation. Here we monitor the effects of leptin on DC migratory capacities, focusing on the intracellular signaling driving cytoskeleton rearrangement. We found that leptin increases immature DC migratory performance both by favoring cytoskeleton dynamics and by up-regulating CCR7 surface expression, thus favoring chemotactic responsiveness. We found that in immature DCs, leptin activates cofilin, favoring the turnover of actin microfilaments, and, by triggering Vav phosphorylation, promotes Rac1 activation. Finally, we found that in immature DCs, leptin up-regulates interleukin-12p70 production on CD40 stimulation and, more importantly, increases their capacity to stimulate activation of autologous CD8(+) T cells. Taken altogether, the findings herein highlight the potential use of leptin as an adjuvant tool in vaccination protocols employing ex vivo-generated autologous DCs.

BACKGROUND AND PURPOSEPropranolol is a vasoactive drug that shows antiangiogenic and antitumour activities in melanoma. However, it is unknown whether these activities are dose-dependent and whether there is a relationship between systemic vascular effects of propranolol and anti-melanoma activity. EXPERIMENTAL APPROACHEffects of increasing doses of propranolol (10, 20, 30 and 40 mg·kg À1 ·day À1 ) on tumour growth were studied in B16F10 melanoma-bearing mice. Histological and biochemical analyses were used to assess propranolol effects on angiogenesis and cancer cell proliferation. Systemic vascular resistance (SVR) was evaluated by measuring cardiac output and arterial BP. KEY RESULTSIn vitro analyses revealed that B16F10 cells expressed β-adrenoceptors, but neither isoprenaline, a β-adrenoceptor agonist, nor the β-blocker propranolol affected cancer cell proliferation. In vivo studies showed that the antitumour efficacy of propranolol follows a U-shaped biphasic dose-response curve. Low doses (10 and 20 mg·kg À1 ·day À1 ) significantly inhibit tumour growth, whereas higher doses are progressively less effective. We also found that high-dose propranolol stimulates tumour arteriogenesis whereas no effect on angiogenesis was observed at any dose. Based on these data and considering that propranolol is a vasoactive drug, we hypothesized that it causes systemic vasoconstriction or vasodilation depending on the dose and thus alters tumour perfusion and growth. Consistent with this hypothesis, we found that propranolol has a biphasic effect on SVR with low and high doses producing vasoconstriction and vasodilation respectively. CONCLUSIONS AND IMPLICATIONSPropranolol inhibits melanoma growth in a U-shaped biphasic manner. A direct relationship exists between SVR and antimelanoma activity.Abbreviations α-SMA, α-smooth muscle actin; CO, cardiac output; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; HR, heart rate; IB4, isolectin B4; SV, stroke volume; SVR, systemic vascular resistance; vWF, Von Willebrand Factor Introduction β-Adrenoceptors are a family of G-protein coupled receptors comprising three subtypes, β 1 , β 2 and β 3 , which act by activating a Gs protein. These receptors play a role in the regulation of peripheral vascular resistance, heart function and airway reactivity, as well as a variety of metabolic and CNS functions. β-Adrenoceptors also regulate cellular processes involved in cancer and angiogenesis and are the molecular target of β-blockers, a class of drugs which inhibits the interaction of catecholamines with β-adrenoceptors.In vitro studies indicate that propranolol, the prototype of β-blockers, reduces melanoma cell proliferation in human and murine melanoma cell lines (Dal Monte et al., 2013;Moretti et al., 2013;Calvani et al., 2015;Wrobel and Le Gal, 2015). Furthermore, the systemic administration of propranolol slows down tumour development in immunodeficient mice transplanted with human melanoma cells (Wrobel and Le Gal, 2015) and inhibits the effects of stress on the growth and metast...

SummaryKawasaki disease (KD) is an acute vasculitis affecting mainly infants and children. Human B cells express Toll-like receptor (TLR)-9, whose natural ligands are unmethylated cytosine-guanine dinucleotide (CpG) motifs characteristic of bacterial DNA. The aim of this study was to clarify the pathogenesis of KD analysing the activation status of peripheral blood mononuclear cells (PBMC), focusing on B lymphocyte activation and functions. Ten patients and 10 age-matched healthy donors were recruited from the Bambino Gesù Hospital of Rome, Italy and enrolled into this study. We determined phenotype profile and immunoglobulin (Ig) production of PBMC from KD patients and age-matched controls. We found that the frequency of CD19 + B lymphocytes and CD19 + /CD86 + activated B lymphocytes from KD patients during the acute phase before therapy was increased significantly. Moreover, B lymphocytes of acute-phase KD patients were more prone to CpG oligodeoxynucleotide (ODN) activation compared with the age-matched controls, as assessed by a significant increase of the number of IgA-secreting cells (SC). In the same patients we found a marked increase of IgM, IgG, interleukin (IL)-6 and tumour necrosis factor (TNF)-a production compared with the control group. In addition, in two convalescent KD patients, conventional treatment with intravenous immunoglobulin (IVIG) restored the normal frequency of CD19 + B cells, the number of IgA-, IgMand IgG-SC and the production of IL-6 and TNF-a. Our findings indicate that the percentages of peripheral B lymphocytes of acute-phase KD patients are increased and are prone to bacterial activation in terms of increased numbers of IgA-SC and increased production of IL-6 and TNF-a inflammatory cytokines. Thus, our data support the hypothesis of an infectious triggering in KD.