Luciana Giordani

Leptin promotes differentiation and survival of human dendritic cells and licenses them for Th1 priming

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.

HIV-1 Nef triggers Vav-mediated signaling pathway leading to functional and morphological differentiation of dendritic cells.

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) ‐κB 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.—Quaranta, M. G., Mattioli, B., Spadaro, F., Straface, E., Giordani, L., Ramoni, C., Malorni, W., Viora, M. HIV‐1 Nef triggers Vav‐mediated signaling pathway leading to functional and morphological differentiation of dendritic cells. FASEB J. 17, 2025–2036 (2003)

Leptin exerts an anti-apoptotic effect on human dendritic cells via the PI3K-Akt signaling pathway

Leptin 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 H2O2‐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, NF‐kB, 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.

IFN-α amplifies human naïve B cell TLR-9-mediated activation and Ig production

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‐α 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‐1β, TNF‐α, 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‐α 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‐α enhances the frequency of CpG ODN‐induced memory B cells. Our results may contribute to clarify the events promoting IFN‐α‐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.

HIV-1 gp120 Accelerates Fas-Mediated Activation-Induced Human Lamina Propria T Cell Apoptosis

Intestinal mucosa represents an important portal of entry of HIV and a site of virus reservoir and active replication. Recently, in HIV patients, an early depletion of intestinal lamina propria T lymphocytes (LPT) has been described. HIV-1 gp120 has been demonstrated to promote apoptosis in noninfected isolated peripheral blood T cells, therefore we investigated whether gp120 modulates apoptosis of normal human intestinal lamina propria T cells. Purified T cells were obtained by immunomagnetic negative selection from human lamina propria mononuclear cells isolated from surgical specimens by enzymatic procedure. Cells were incubated with or without recombinant gp120 (10 μg/ml) and cultured either in the absence of any stimulus or in the presence of plate-bound anti-CD3 Ab (OKT3) or soluble anti-CD2 Ab (T112 + T113). Apoptosis was assessed by flow cytometric analysis after propidium iodide staining. We demonstrated that preincubation of normal LPT cells with HIV-1 gp120 accelerates the apoptosis observed during CD2-pathway stimulation of LPT cells. This process is mediated by Fas/Fas ligand interaction and related to an increased induction of Fas ligand mRNA by gp120. Therefore HIV-1 gp120 could contribute to the depletion of noninfected LPT cells inducing a premature cell death.

HIV-1 Nef equips dendritic cells to reduce survival and function of CD8+ T cells: a mechanism of immune evasion

The accessory HIV‐1 Nef protein is a crucial determinant for viral replication and pathogenesis. During HIV infection, loss of immune control in the setting of a strong and broad HIV‐specific T‐lymphocyte response, leads to a lethal outcome through AIDS. Moreover, dysfunction of dendritic cells (DCs) may contribute to the immune suppression associated with AIDS progression. We recently demonstrated that exogenous Nef selectively activates immature DCs manipulating their phenotypical, morphological, and functional developmental program. Here, we tracked whether Nef, targeting DCs, could be involved in the dysregulation of CD8+ T cell responses. We found that Nef inhibits the capacity of DCs to prime alloreactive CD8+ T cell responses down‐regulating their proliferation and functional competence. This coincides with the induction of CD8+ T cell apoptosis. Nef oversees apoptotic killing of CD8+ T cells up‐regulating TNF‐α and FasL production by DCs and interfering with the death receptor pathway in CD8+ T cells and thus activating caspase 8. Our findings suggest that Nef may contribute to the immune evasion associated with HIV‐1 infection, subverting DC biology. This may help explain the pleiotropic function that Nef plays during infection and makes this protein an attractive target for preventive and therapeutic intervention.

Leptin as an immunological adjuvant: enhanced migratory and CD8+ T cell stimulatory capacity of human dendritic cells exposed to leptin

Leptin is an adipocyte‐derived hor‐ mone/cytokine that links nutrition, metabolism, and immune homeostasis and is endowed to modulate several immune responses. We previously demon strated that both immature and mature human den dritic 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 sig naling driving cytoskeleton rearrangement. We found that leptin increases immature DC migratory perfor mance 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 phos phorylation, promotes Rac1 activation. Finally, we found that in immature DCs, leptin up‐regulates inter‐ leukin‐12p70 production on CD40 stimulation and, more importantly, increases their capacity to stimulate activation of autologous CD8+ T cells. Taken alto gether, the findings herein highlight the potential use of leptin as an adjuvant tool in vaccination protocols employing ex vivo‐generated autologous DCs.— Mat‐ tioli, B., Straface, E., Matarrese, P., Quaranta, M. G., Giordani, L., Malorni, W., Viora, M. Leptin as an immunological adjuvant: enhanced migratory and CD8+T cell stimulatory capacity of human dendritic cells exposed to leptin. FASEB J. 22, 2012–2022 (2008)

The immunoregulatory effects of HIV-1 Nef on dendritic cells and the pathogenesis of AIDS

Dendritic cells (DC) play a crucial role in the generation and regulation of immunity, and their interaction with HIV is relevant in the pathogenesis of AIDS favoring both the initial establishment and spread of the infection and the development of antiviral immunity. HIV‐1 Nef is an essential factor for efficient viral replication and pathogenesis, and several studies have been addressed to assess the possible influence of endogenous or exogenous Nef on DC biology. Our findings and other reported data described in this review demonstrate that Nef subverts DC biology interfering with phenotypical, morphological, and functional DC developmental programs, thus representing a viral tool underlying AIDS pathogenesis. This review provides an overview on the mechanism by which Nef, hijacking DC functional activity, may favor both the replication of HIV‐1 and the escape from immune surveillance. Overall, the findings described here may contribute to the understanding of Nef function, mechanism of action, and cellular partners. Further elucidation of genes induced through Nef signaling in DC could reveal pathways used by DC to drive HIV spread and will be critical to identify therapeutic strategies to bias the DC system toward activation of antiviral immunity instead of facilitating virus dissemination.—Quaranta, M. G., Mattioli, B., Giordani, L., Viora, M. The immunoregulatory effects of HIV‐1 Nef on dendritic cells and the pathogenesis of AIDS. FASEB J. 20, 2198–2208 (2006)

HIV-1 Nef impairs the dynamic of DC/NK crosstalk: different outcome of CD56dim and CD56bright NK cell subsets

Dendritic cells (DCs) and natural killer (NK) cells are essential components of the innate immunity and play a critical role in the first phase of host defense against infection. Interactions between DCs and NK cells have been demonstrated in a variety of settings, with evidence emerging of complex bidirectional crosstalk between the two cell types. The accessory HIV‐1 Nef protein is a crucial determinant for viral replication and pathogenesis. We previously demon‐strated that Nef, hijacking DC functional activity, subverts the DC arm of immune response to escape the adaptive immune attack. Here, we monitor the effect of Nef on the outcome of the innate immune response, focusing on the impact of Nef on DC/NK crosstalk. We demonstrate that Nef up‐regulates the ability of DCs to stimulate the immunoregulatory NK cells (CD56bright) as assessed by the activated phenotype, up‐regulation of their proliferative response and INF‐γ release. On the other hand, Nef‐pulsed DCs inhibit cytotoxic NK cells (CD56dim), as assessed by the reduced HLA‐DR surface expression, reduced proliferation and cytotoxic activity. Moreover, in the presence of Nef‐pulsed DCs, we found a significant up‐regulation of TNF‐α secretion and a significant reduction of IL‐10, GM‐CSF, MIP‐1α and RANTES secretion. Our findings suggest that the Nef‐induced dysregulation in the DC/NK cell crosstalk may represent a potential mechanism through which HIV escapes innate immune surveillance.—Quaranta, M.G., Napolitano, A., Sanchez, M., Giordani, L., Mattioli, B., Viora, M. HIV‐1 Nef impairs the dynamic of DC/NK crosstalk: different outcome of CD56dim and CD56bright NK cell subsets. FASEB J. 21, 2323–2334 (2007)

Does Oxidative Stress Play a Critical Role in Cardiovascular Complications of Kawasaki Disease

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.