Lactoferrin differently modulates the inflammatory response in epithelial models mimicking human inflammatory and infectious diseases.

Conflicting data are reported on pro- or anti-inflammatory activity of bovine lactoferrin (bLf) in different cell models as phagocytes or epithelial cell lines infected by bacteria. Here we evaluated the bLf effect on epithelial models mimicking two human pathologies characterized by inflammation and infection with specific bacterial species. Primary bronchial epithelium from a cystic fibrosis (CF) patient and differentiated intestinal epithelial cells were infected with Pseudomonas aeruginosa LESB58 isolated from a CF patient and Adherent-Invasive Escherichia coli LF82 isolated from a Crohn's disease patient. Surprisingly, bLf significantly reduced the intracellular bacterial survival, but differently modulated the inflammatory response. These data lead us to hypothesize that bLf differentially acts depending on the epithelial model and infecting pathogen. To verify this hypothesis, we explored whether bLf could modulate ferroportin (Fpn), the only known cellular iron exporter from cells, that, by lowering the intracellular iron level, determines a non permissive environment for intracellular pathogens. Here, for the first time, we describe the bLf ability to up-regulate Fpn protein in infected epithelial models. Our data suggest that the mechanism underlying the bLf modulating activity on inflammatory response in epithelial cells is complex and the bLf involvement in modulating cellular iron homeostasis should be taken into account.

LF immunomodulatory strategies: mastering bacterial endotoxin.

Lactoferrin (LF), an iron-binding glycoprotein expressed in most biological fluids, represents a major component of mammalian innate immune system. The multiple activities of LF rely not only on its capacity to bind iron but also to interact with molecular and cellular components of both the host and pathogens. LF can bind and sequester lipopolysaccharide thus preventing proinflammatory pathway activation, sepsis, and tissue damage. However, the interplay between LF and lipopolysaccharide is complex and may lead to different outcomes including both the suppression of inflammatory response and immune activation. Understanding the molecular basis and the functional consequences of this complex interaction is critically relevant in the development of LF-based therapeutic interventions in humans.

Immunoregulatory role of lactoferrin-lipopolysaccharide interactions.

Lactoferrin (Lf) is a mammalian exclusive protein widely distributed in milk and exocrine secretions exhibiting multifunctional properties. Many of the proven or proposed functions of Lf, apart from its iron binding activity, depend on its capacity to bind to other macromolecules. Lf can bind and sequester lipopolysaccharide (LPS), thus preventing pro-inflammatory pathway activation, sepsis and tissue damage. However, the interplay between Lf and LPS is complex, and may result in different outcomes, including both suppression of the inflammatory response and immune activation. These findings are critically relevant in the development of Lf-based therapeutic interventions in humans. Understanding the molecular basis and functional consequences of Lf-LPS interaction will provide insights for determining its role in health and disease.

Role of endogenous interferon and LPS in the immunomodulatory effects of bovine lactoferrin in murine peritoneal macrophages.

Lactoferrin (Lf) plays an important role in host defense against infection and excessive inflammation. Although the mechanisms underlying its immunomodulatory properties have not been fully elucidated yet, recent evidence suggests that some of these effects may be related to its capacity to form complexes with LPS. We report that the culture of resting mouse peritoneal macrophages (PM) with bovine Lf (bLf), prior to infection with the vesicular stomatitis virus (VSV), resulted in a significant reduction of virus yield with respect to control cultures. The antiviral activity of bLF was related to its capacity of inducing IFN-alpha/beta expression, which in turn inhibited VSV replication. Indeed, the accumulation of IFN-beta but not of IFNalpha(1-2) transcripts was up-modulated markedly early after bLf addition. Furthermore, bLf did not exert any antiviral activity in the presence of neutralizing antibodies to IFN-alpha/beta in PM from wild-type mice, as well as in PM from mice genetically defective for the response to IFN. The antiviral activity of bLf relied on its intrinsic capacity to bind LPS, as this protein did not induce IFN expression in PM from LPS-hyporesponsive mice. It is interesting that this LPS-binding property was dispensable for the production of TNF-alpha, which also occurred in LPS-hyporesponsive mice. Overall, these results indicate that some of the immunomodulatory effects ascribed to Lf may be related to its capacity to favor Type I IFN expression and argue in favor of an important role of the LPS-binding feature and TLR4 in some of the effects ascribed to this molecule.

Direct and Intestinal Epithelial Cell-Mediated Effects of TLR8 Triggering on Human Dendritic Cells, CD14+CD16+ Monocytes and γδ T Lymphocytes.

Toll-like receptor (TLR)7/8 plays a crucial role in host recognition/response to viruses and its mucosal expression directly correlates with intestinal inflammation. The aim of this study was to investigate the role of TLR7/8 stimulation of intestinal epithelium in shaping the phenotype and functions of innate immunity cell subsets, and to define direct and/or epithelial cell-mediated mechanisms of the TLR7/8 agonist R848 immunomodulatory activity. We describe novel, TLR8-mediated, pro- and anti-inflammatory effects of R848 on ex vivo cultured human blood monocytes and γδ T lymphocytes, either induced by direct immune cell stimulation or mediated by intestinal epithelial cells (IEC). Apical stimulation with R848 led to its transport across normal polarized epithelial cell monolayer and resulted in the inhibition of monocyte differentiation toward immunostimulatory dendritic cells and Th1 type response. Furthermore, γδ T lymphocyte activation was promoted following direct exposure of these cells to the agonist. Conversely, a selective enrichment of the CD14+CD16+ monocyte subpopulation was observed, which required a CCL2-mediated inflammatory response of normal epithelial cells to R848. Of note, a TLR-mediated activation of control γδ T lymphocytes was promoted by inflamed intestinal epithelium from active Crohn's disease patients. This study unravels a novel regulatory mechanism linking the activation of the TLR8 pathway in IEC to the monocyte-mediated inflammatory response, and highlights the capacity of the TLR7/8 agonist R848 to directly enhance the activation of γδ T lymphocytes. Overall these results expand the range of cell targets and immune responses controlled by TLR8 triggering that may contribute to the antiviral response, to chronic inflammation, as well as to the adjuvant activity of TLR8 agonists, highlighting the role of intestinal epithelium microenvironment in shaping TLR agonist-induced responses.

IL-2 induces expression and secretion of IFN-gamma in murine peritoneal macrophages.

We investigated the effect of interleukin (IL)-2, a T cell growth factor capable of activating certain macrophage functions, on interferon (IFN)-gamma expression in resting mouse peritoneal macrophages (PM). IL-2 addition to PM from different mouse strains up-modulated IFN-gamma mRNA and protein secretion. It is notable that endogenous type I and II IFNs did not play any role in the IL-2-mediated effect, as comparable levels of secreted IFN-gamma were observed upon IL-2 stimulation of PM from deficient mice. In contrast, endogenous IFN-gamma was requested for the IL-12-induced IFN-gamma production. It is interesting that blocking of each component of the IL-2 receptor (IL-2R) by neutralizing antibodies almost completely abolished IL-2-induced IFN-gamma production, suggesting that all IL-2R chains contribute to the PM biological response to IL-2. The simultaneous treatment of PM with IL-2 and IL-12 resulted in a higher IFN-gamma secretion with respect to that obtained upon treatment with IL-2 or IL-12 alone. It is notable that IFN-gamma protein was expressed intracellularly in the majority of cells exhibiting a macrophage phenotype (i.e., F4/80+) and was secreted upon IL-2 stimulation. Overall, these findings demonstrate that IL-2 regulates at different levels IFN-gamma expression in macrophages, highlighting the crucial role of these cells and their regulated responsiveness to key cytokines in the cross-talk between innate and adaptive immunity.

Bovine Lactoferrin-Induced CCL1 Expression Involves Distinct Receptors in Monocyte-Derived Dendritic Cells and Their Monocyte Precursors.

Lactoferrin (LF) exhibits a wide range of immunomodulatory activities including modulation of cytokine and chemokine secretion. In this study, we demonstrate that bovine LF (bLF) up-modulates, in a concentration- and time-dependent manner, CCL1 secretion in monocytes (Mo) at the early stage of differentiation toward dendritic cells (DCs), and in fully differentiated immature Mo-derived DCs (MoDCs). In both cell types, up-modulation of CCL1 secretion is an early event following bLF-mediated enhanced accumulation of CCL1 transcripts. Notably, bLF-mediated up-regulation of CCL1 involves the engagement of distinct surface receptors in MoDCs and their Mo precursors. We show that bLF-mediated engagement of CD36 contributes to CCL1 induction in differentiating Mo. Conversely, toll-like receptor (TLR)2 blocking markedly reduces bLF-induced CCL1 production in MoDCs. These findings add further evidence for cell-specific differential responses elicited by bLF through the engagement of distinct TLRs and surface receptors. Furthermore, the different responses observed at early and late stages of Mo differentiation towards DCs may be relevant in mediating bLF effects in specific body districts, where these cell types may be differently represented in physiopathological conditions.

Bovine lactoferrin counteracts Toll-like receptor mediated activation signals in antigen presenting cells.

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.

Reciprocal interactions between lactoferrin and bacterial endotoxins and their role in the regulation of the immune response.

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.

Immunomodulatory effects of lactoferrin on antigen presenting cells.

Lactoferrin (Lf) is an 80 kDa iron-binding protein of the transferrin family that is abundantly expressed in most biological fluids. It is now recognized that this glycoprotein is a key element in the mammalian immune system, playing an important role in host defence against infection and excessive inflammation. Although the mechanisms underlying Lf immunomodulatory properties have not been fully elucidated yet, evidence indicates that the capacity of this molecule to directly interact with antigen presenting cells (APCs), i.e. monocytes/macrophages and dendritic cells (DCs), may play a critical role. At the cellular level, Lf modulates important aspects of APC biology, including migration and cell activation, whereas at the molecular level it affects expression of soluble immune mediators, such as cytokines, chemokines and other effector molecules, thus contributing to the regulation of inflammation and immunity. While the iron-binding property was originally believed to be solely responsible for the plethora of host defence activities ascribed to Lf, it is now known that other mechanisms contribute to the broad spectrum of anti-infective and anti-inflammatory properties of this protein. Recent results suggest that at least some of the immunomodulatory effects of Lf rely on its capacity to form complexes with lipopolysaccharide (LPS). This review focuses on the effects of Lf on APC biology and function, highlighting known and putative mechanisms that underlie Lf immunomodulatory effects. The importance of LPS-binding capacity of Lf and LPS receptors, as well as of Lf-induced type 1 interferon (IFN) expression in some of these effects is also discussed.

Role of the cytokine environment and cytokine receptor expression on the generation of functionally distinct dendritic cells from human monocytes.

Myeloid dendritic cells (DC) and macrophages evolve from a common precursor. However, factors controlling monocyte differentiation toward DC or macrophages are poorly defined. We report that the surface density of the GM-CSF receptor (GM-CSFR) alpha subunit in human peripheral blood monocytes varies among donors. Although no correlation was found between the extent of GM-CSFR and monocyte differentiation into DC driven by GM-CSF and IL-4, GM-CSFR expression strongly influenced the generation of CD1a(+) dendritic-like cells in the absence of IL-4. CD1a(+) cells generated in the presence of GM-CSF express CD40, CD80, MHC class I and II, DC-SIGN, MR, CCR5, and partially retain CD14 expression. Interestingly, they spontaneously induce the expansion of CD4(+) and CD8(+) allogeneic T lymphocytes producing IFN-gamma, and migrate toward CCL4 and CCL19. Upon stimulation with TLR ligands, they acquire the phenotypic features of mature DC. In contrast, the allostimulatory capacity is not further increased upon LPS activation. However, by blocking LPS-induced IL-10, a higher T cell proliferative response and IL-12 production were observed. Interestingly, IL-23 secretion was not affected by endogenous IL-10. These results highlight the importance of GM-CSFR expression in monocytes for cytokine-induced DC generation and point to GM-CSF as a direct player in the generation of functionally distinct DC.

P-glycoprotein-actin association through ERM family proteins: a role in P-glycoprotein function in human cells of lymphoid origin.

P-glycoprotein is a 170-kd glycosylated transmembrane protein, expressed in a variety of human cells and belonging to the adenosine triphosphate-binding cassette transporter family, whose membrane expression is functionally associated with the multidrug resistance phenotype. However, the mechanisms underlying the regulation of P-glycoprotein functions remain unclear. On the basis of some evidence suggesting P-glycoprotein-actin cytoskeleton interaction, this study investigated the association of P-glycoprotein with ezrin, radixin, and moesin, a class of proteins that cross-link actin filaments with plasma membrane in a human cell line of lymphoid origin and that have been shown to link other ion-pump-related proteins. To this purpose, a multidrug-resistant variant of CCRF-CEM cells (CEM-VBL100) was used as a model to investigate the following: (1) the cellular localizations of P-glycoprotein and ezrin, radixin, and moesin and their molecular associations; and (2) the effects of ezrin, radixin, and moesin antisense oligonucleotides on multidrug resistance and P-glycoprotein function. The results showed that: (1) P-glycoprotein colocalized and coimmunoprecipitated with ezrin, radixin, and moesin; and (2) treatment with antisense oligonucleotides for ezrin, radixin, and moesin restored drug susceptibility consistently with inhibition of both drug efflux and actin-P-glycoprotein association and induction of cellular redistribution of P-glycoprotein. These data suggest that P-glycoprotein association with the actin cytoskeleton through ezrin, radixin, and moesin is key in conferring to human lymphoid cells a multidrug resistance phenotype. Strategies aimed at inhibiting P-glycoprotein-actin association may be helpful in increasing the efficiency of both antitumor and antiviral therapies.