Lactoferrin (LF), a key element in mammalian immune system, plays pivotal roles in host defence against infection and excessive inflammation. Its protective effects range from direct antimicrobial activities against a large panel of microbes, including bacteria, viruses, fungi and parasites, to antinflammatory and anticancer activities. In this study, we show that monocyte-derived dendritic cells (MD-DCs) generated in the presence of bovine LF (bLF) fail to undergo activation by up-modulating CD83, co-stimulatory and major histocompatibility complex molecules, and cytokine/chemokine secretion. Moreover, these cells are weak activators of T cell proliferation and retain antigen uptake activity. Consistent with an impaired maturation, bLF-MD-DC primed T lymphocytes exhibit a functional unresponsiveness characterized by reduced expression of CD154 and impaired expression of IFN-γ and IL-2. The observed imunosuppressive effects correlate with an increased expression of molecules with negative regulatory functions (i.e. immunoglobulin-like transcript 3 and programmed death ligand 1), indoleamine 2,3-dioxygenase, and suppressor of cytokine signaling-3. Interestingly, bLF-MD-DCs produce IL-6 and exhibit constitutive signal transducer and activator of transcription 3 activation. Conversely, bLF exposure of already differentiated MD-DCs completely fails to induce IL-6, and partially inhibits Toll-like receptor (TLR) agonist-induced activation. Cell-specific differences in bLF internalization likely account for the distinct response elicited by bLF in monocytes versus immature DCs, providing a mechanistic base for its multiple effects. These results indicate that bLF exerts a potent anti-inflammatory activity by skewing monocyte differentiation into DCs with impaired capacity to undergo activation and to promote Th1 responses. Overall, these bLF-mediated effects may represent a strategy to block excessive DC activation upon TLR-induced inflammation, adding further evidence for a critical role of bLF in directing host immune function.

Background

The advent of Systems Biology has been accompanied by the blooming of pathway databases. Currently pathways are defined generically with respect to the organ or cell type where a reaction takes place. The cell type specificity of the reactions is the foundation of immunological research, and capturing this specificity is of paramount importance when using pathway-based analyses to decipher complex immunological datasets. Here, we present DC-ATLAS, a novel and versatile resource for the interpretation of high-throughput data generated perturbing the signaling network of dendritic cells (DCs).

Results

Pathways are annotated using a novel data model, the Biological Connection Markup Language (BCML), a SBGN-compliant data format developed to store the large amount of information collected. The application of DC-ATLAS to pathway-based analysis of the transcriptional program of DCs stimulated with agonists of the toll-like receptor family allows an integrated description of the flow of information from the cellular sensors to the functional outcome, capturing the temporal series of activation events by grouping sets of reactions that occur at different time points in well-defined functional modules.

Conclusions

The initiative significantly improves our understanding of DC biology and regulatory networks. Developing a systems biology approach for immune system holds the promise of translating knowledge on the immune system into more successful immunotherapy strategies.

Lactoferrin (Lf), an iron-binding glycoprotein expressed in most biological fluids, represents a major component of the mammalian innate immune system. Lf’s multiple activities rely not only on its capacity to bind iron, but also to interact with molecular and cellular components of both host and pathogens. Lf can bind and sequester lipopolysaccharide (LPS), thus preventing pro-inflammatory pathway activation, sepsis and tissue damage. However, Lf-bound LPS may retain the capacity to induce cell activation via Toll-like receptor 4-dependent and -independent mechanisms. This review discusses the complex interplay between Lf and LPS and its relevance in the regulation of the immune response.

Exposure to aldrithiol-2-inactivated human immunodeficiency virus type 1 or gp120, but not gp41, triggered alpha interferon (IFN-α), CC chemokine ligand 2 (CCL2), CCL3, and CCL4 production in human plasmacytoid dendritic cells (DCs) but not in myeloid DCs (M-DCs) or monocyte-derived DCs from the same donors. The nonresponsiveness of M-DCs for IFN-α/β production was a general feature specific to these cells, as they also failed to produce it in response to inactivated influenza virus, poly(I-C), lipopolysaccharide, Staphylococcus aureus Cowans I, or CD40L. The different capacities of circulating DC subsets to produce immune mediators in response to most stimuli argue for a different role for these cells in the regulation of innate immunity to pathogens.

Dendritic cells (DCs) play a crucial role in bridging innate and acquired immune responses to pathogens. In human immunodeficiency virus type 1 (HIV-1) infection, immature DCs (iDCs) are also main targets for HIV-1 at the mucosal level. In this study, we evaluated the effects of HIV-1-DC interactions on the maturation and functional activity of these cells. Exposure of human monocyte-derived iDCs to either aldrithiol-2-inactivated HIV-1 or gp120 led to an upmodulation of activation markers indicative of functional maturation. Despite their phenotype, these cells retained antigen uptake capacity and showed an impaired ability to secrete cytokines or chemokines and to induce T-cell proliferation. Although gp120 did not interfere with DC differentiation, the capacity of these cells to produce interleukin-12 (IL-12) upon maturation was markedly reduced. Likewise, iDCs stimulated by classical maturation factors in the presence of gp120 lacked allostimulatory capacity and did not produce IL-12, in spite of their phenotype typical of activated DCs. Exogenous addition of IL-12 restores the allostimulatory capacity of gp120-exposed DCs. The finding that gp120 induces abnormal maturation of DCs linked to profound suppression of their activities unravels a novel mechanism by which HIV can lead to immune dysfunction in AIDS patients.

Jurkat T-cell clones, stably expressing the human immunodeficiency virus type 1 (HIV-1) Vpr protein, exhibited an impaired susceptibility to HIV-1 infection. A marked down-modulation of surface CD4 receptors was detected in Vpr-expressing clones with respect to control cells. Likewise, a reduced CD4 expression was also observed in parental Jurkat cells infected with wild-type but not with Vpr-mutant HIV-1. Notably, Vpr-expressing clones were fully susceptible to infection with a vesicular stomatitis virus G protein-pseudotyped HIV-1 virus, indicating that a block at the level of viral entry was responsible for the inhibition of viral replication. The effect exerted by Vpr on HIV replication and CD4 expression suggests that this protein can regulate both the establishment of a productive HIV-1 infection and CD4-mediated T-cell functions.

We investigated the role of the constitutive nitric oxide (NO) in the expression of interferon (IFN) genes in mouse peritoneal macrophages (PM). The treatment of PM with l-arginine-NG-amine (AA), a potent inhibitor of NO-producing enzymes, resulted in a marked accumulation of IFN-α4 mRNA and, to a minor extent, of IFN-β mRNA. In contrast, the expression of IFN-γ mRNA, as well as tumor necrosis factor alpha and interleukin-6 mRNA, was not affected. Furthermore, a remarkable increase in the expression of the IFN regulating factor 1 (IRF-1), but not of IRF-2, mRNA was detected in AA-treated PM. To investigate whether the AA-induced activation of the IFN system correlates with the production and antiviral activity of IFN, the extent of encephalomyocarditis virus (EMCV) replication was monitored in AA-treated PM with respect to control cultures. AA treatment strongly inhibited, in a dose-dependent manner, EMCV yields in PM. Likewise, similar results were obtained by the addition of the NO-scavenger carboxyphenyl-tetramethylimidazoline-oxyl-oxide. In addition, inhibition of NO synthesis by NG-mono-methyl-l-arginine in PM strongly decreased virus replication in coculture of PM and EMCV-infected L929 cells, whereas no antiviral effect was observed in L929 cells alone. Moreover, the AA-mediated antiviral activity was abrogated in the presence of antibody to IFN-α/β, whereas antibody to IFN-γ was completely ineffective. Taken together, these results indicate that low levels of NO, constitutively released by resting PM, negatively regulate the expression and activity of IFN-α/β in PM. We suggest that NO acts as a homeostatic agent in the regulation of IFN pathway expression in macrophages.