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.

The presence of HIV-1 in the intestinal mucosa of AIDS patients has been reported and human intestinal lamina propria lymphocytes (LPL) have been proposed as important targets for HIV-1 infection. However, little information is available concerning the permissiveness of human intestinal CD4+ T lymphocytes to HIV-1 infection. Here, we show that human LPL, in contrast to autologous peripheral blood lymphocytes (PBL), are permissive to both X4 T-tropic and R5 M-tropic strains of HIV-1, as well as to clinical isolates, in the absence of exogenous stimuli. Flow cytometry showed that the vast majority of T LPL were CD45RO+ and CD69+, and that CD4+ T LPL highly expressed CC chemokine receptor 5 (CCR5) as compared to PBL, while CX chemokine receptor 4 was equally expressed on LPL and PBL. Exogenous RANTES and macrophage inflammatory protein-1alpha (natural CCR5 ligands) virtually abolished the entry of the R5 M-tropic strain HIV-1 into human LPL. Thus, we infer that human intestinal CD4+ T lymphocytes are naturally susceptible to HIV-1 infection, due to their physiological state of activation and to marked expression of HIV-1 coreceptors, independently of the route of primary (either mucosal or parental) infection and the shifts of the virus phenotype occurring during the course of AIDS.

Cytotoxic necrotizing factor 1 (CNF1) is a protein toxin produced by some pathogenic strains of Escherichia coli that specifically activates Rho, Rac, and Cdc42 GTPases. We previously reported that this toxin prevents the ultraviolet-Binduced apoptosis in epithelial cells, with a mechanism that remained to be defined. In this work, we show that the proteasomal degradation of the Rho GTPase is necessary to achieve cell death protection, because inhibition of Rho degradation abolishes the prosurvival activity of CNF1. We hypothesize that Rho inactivation allows the activity of Rac to become dominant. This in turn leads to stimulation of the phosphoinositide 3-kinase/Akt/I B kinase/nuclear factor-B prosurvival pathway and to a remarkable modification in the architecture of the mitochondrial network, mainly consisting in the appearance of elongated and interconnected mitochondria. Importantly, we found that Bcl-2 silencing reduces the ability of CNF1 to protect cells against apoptosis and that it also prevents the CNF1-induced mitochondrial changes. It is worth noting that the ability of a bacterial toxin to induce such a remodeling of the mitochondrial network is herein reported for the first time. The possible pathophysiological relevance of this finding is discussed. INTRODUCTIONToday, it is largely acknowledged that apoptosis, besides being an evolutionary conserved form of cell death that plays a pivotal role during development, morphogenesis, and cell homeostasis, is also critically implied in a constantly growing number of diseases (Fadeel and Orrenius, 2005). In fact, apoptosis can be regarded as a widespread strategy exploited by pathogenic bacteria to favor their own survival or spreading in the host , often by producing protein toxins that mediate their long-range cross-talk with host cells. In this context, we have previously reported the ability of a protein toxin from Escherichia coli, namely the cytotoxic necrotizing factor 1 (CNF1), to prevent the ultraviolet-B (UVB)-induced apoptosis and to increase the expression of antiapoptotic Bcl-2 family proteins (Fiorentini et al., 1998). The precise mechanism by which CNF1 allows cells to survive, however, is not yet defined.CNF1 is a protein toxin produced by some pathogenic strains of E. coli mainly involved in extraintestinal infections (Landraud et al., 2000). In eukaryotic cells, CNF1 binds to its receptor, reported to be the receptor of laminin , and it is endocytosed and released into the cytoplasm by an acidic-dependent mechanism (Contamin et al., 2000). Once in the cytoplasm, CNF1 exerts its enzymatic activity that is represented by deamidation of a pivotal glutamine residue of the guanosine triphosphate (GTP)-binding proteins Rho, Rac, and Cdc42 (glutamine 63 of Rho or glutamine 61 of Rac and Cdc42), giving rise to a glutamic acid (Flatau et al., 1997;Schmidt et al., 1997;Lerm et al., 1999). The glutamine residue modified by CNF1 lies in the switch 2 domain of Rho proteins, which is involved in GTP hydrolysis; thus, the modification exerted by CNF1...

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.

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