Glucocorticoid-Induced Leucine Zipper Protein Controls Macropinocytosis in Dendritic Cells.

Ag sampling is a key process in dendritic cell (DC) biology. DCs use constitutive macropinocytosis, receptor-mediated endocytosis, and phagocytosis to capture exogenous Ags for presentation to T cells. We investigated the mechanisms that regulate Ag uptake by DCs in the steady-state and after a short-term LPS exposure in vitro and in vivo. We show that the glucocorticoid-induced leucine zipper protein (GILZ), already known to regulate effector versus regulatory T cell activation by DCs, selectively limits macropinocytosis, but not receptor-mediated phagocytosis, in immature and recently activated DCs. In vivo, the GILZ-mediated inhibition of Ag uptake is restricted to the CD8α+ DC subset, which expresses the highest GILZ level among splenic DC subsets. In recently activated DCs, we further establish that GILZ limits p38 MAPK phosphorylation, providing a possible mechanism for GILZ-mediated macropinocytosis control. Finally, our results demonstrate that the modulation of Ag uptake by GILZ does not result in altered Ag presentation to CD4 T cells but impacts the efficiency of cross-presentation to CD8 T cells. Altogether, our results identify GILZ as an endogenous inhibitor of macropinocytosis in DCs, the action of which contributes to the fine-tuning of Ag cross-presentation.

Phosphoinositides in phagocytosis and macropinocytosis.

Professional phagocytes provide immunoprotection and aid in the maintenance of tissue homeostasis. They perform these tasks by recognizing, engulfing and eliminating pathogens and endogenous cell debris. Here, we examine the paramount role played by phosphoinositides in phagocytosis and macropinocytosis, two major endocytic routes that mediate the uptake of particulate and fluid matter, respectively. We analyze accumulating literature describing the molecular mechanisms whereby phosphoinositides translate environmental cues into the complex, sophisticated responses that underlie the phagocytic and macropinocytic responses. In addition, we exemplify virulence strategies involving modulation of host cell phosphoinositide signaling that are employed by bacteria to undermine immunity. This article is part of a Special Issue entitled Phosphoinositides.

Inflammatory stimuli reprogram macrophage phagocytosis to macropinocytosis for the rapid elimination of pathogens.

Following an infectious challenge, macrophages have to be activated in order to allow efficient clearance of infectious pathogens, but how macrophage activation is coupled to increased clearance remains largely unknown. We here describe that inflammatory stimuli induced the reprogramming of the macrophage endocytic machinery from receptor-mediated phagocytosis to macropinocytosis, allowing the rapid transfer of internalized cargo to lysosomes in a receptor-independent manner. Reprogramming occurred through protein kinase C-mediated phosphorylation of the macrophage protein coronin 1, thereby activating phosphoinositol (PI)-3-kinase activity necessary for macropinocytic uptake. Expression of a phosphomimetic form of coronin 1 was sufficient to induce PI3-kinase activation and macropinocytosis even in the absence of inflammatory stimuli. Together these results suggest a hitherto unknown mechanism to regulate the internalization and degradation of infectious material during inflammation.

Human metapneumovirus SH and G glycoproteins inhibit macropinocytosis-mediated entry into human dendritic cells and reduce CD4+ T cell activation.

UNLABELLED: Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We used gene-deletion viruses to evaluate the role of the attachment G and small hydrophobic SH glycoproteins on virus uptake by primary human monocyte-derived dendritic cells (MDDC) in vitro and on subsequent MDDC maturation and activation of autologous T cells. HMPV with deletion of G and SH (ΔSHG) exhibited increased infectivity but had little effect on MDDC maturation. However, MDDC stimulated with ΔSHG induced increased proliferation of autologous Th1-polarized CD4(+) T cells. This effect was independent of virus replication. Increased T cell proliferation was strictly dependent on contact between virus-stimulated MDDC and CD4(+) T cells. Confocal microscopy revealed that deletion of SH and G was associated with an increased number of immunological synapses between memory CD4(+) T cells and virus-stimulated MDDC. Uptake of HMPV by MDDC was found to be primarily by macropinocytosis. Uptake of wild-type (WT) virus was reduced compared to that of ΔSHG, indicative of inhibition by the SH and G glycoproteins. In addition, DC-SIGN-mediated endocytosis provided a minor alternative pathway that depended on SH and/or G and thus operated only for WT. Altogether, our results show that SH and G glycoproteins reduce the ability of HMPV to be internalized by MDDC, resulting in a reduced ability of the HMPV-stimulated MDDC to activate CD4(+) T cells. This study describes a previously unknown mechanism of virus immune evasion. IMPORTANCE: Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We found that HMPV attachment G and small hydrophobic SH glycoproteins reduce the ability of HMPV to be internalized by macropinocytosis into human dendritic cells (DC). This results in a reduced ability of the HMPV-stimulated DC to activate Th1-polarized CD4(+) T cells. These results contribute to a better understanding of the nature of incomplete protection against this important human respiratory virus, provide new information on the entry of HMPV into human cells, and describe a new mechanism of virus immune evasion.

[THE DYNAMICS OF IMMUNOLOGICAL RESULTS OF PATIENTS WITH T-CELL SKIN LYMPHOMAS AND PSORIASIS BY THE THERAPY OF ACTIVATION MECHANISMS SANOGENESIS METHODS].

The therapy T-cell skin lymphoma and psoriasis by the application of activation mechanisms sanogenesis methods, such as: original--a treatment plasmapheresis, a standard heparin infusion; used for the first time--wobenzym; solutions of acid acetic food and sodium bicarbonate; known--the basic sanitations of concomitant diseases, photopheresis caused remissions in 79.6% patients with different stages T-cell skin lymphoma (observed over an 8-year span), and in 67% of patients with psoriasis (observed over an 6-year span). Depuration reactions (phagocytosis, pinocytosis, toxin neutralization) has been activated by detoxication of treatment plasmapheresis and heparin infusions. The topical therapy with wobenzym, solutions of acid acetic food and sodium bicarbonate renewed natural immune barrier of skin. Basic therapy of concomitant diseases enhanced of patient state of health and mobilized compensatory resources. Photopheresis initiated autoimmunization processes by malignant CD4+ lymphocytes. When remission was achieved, the parameters of cellular and humoral immunity returned to normal levels, or the parameters made worse in the absence of remission.

Temporal transcriptional response during infection of type II alveolar epithelial cells with Francisella tularensis live vaccine strain (LVS) supports a general host suppression and bacterial uptake by macropinocytosis.

Pneumonic tularemia is caused by inhalation of Francisella tularensis, one of the most infectious microbes known. We wanted to study the kinetics of the initial and early interactions between bacterium and host cells in the lung. To do this, we examined the infection of A549 airway epithelial cells with the live vaccine strain (LVS) of F. tularensis. A549 cells were infected and analyzed for global transcriptional response at multiple time points up to 16 h following infection. At 15 min and 2 h, a strong transcriptional response was observed including cytoskeletal rearrangement, intracellular transport, and interferon signaling. However, at later time points (6 and 16 h), very little differential gene expression was observed, indicating a general suppression of the host response consistent with other reported cell lines and murine tissues. Genes for macropinocytosis and actin/cytoskeleton rearrangement were highly up-regulated and common to the 15 min and 2 h time points, suggesting the use of this method for bacterial entry into cells. We demonstrate macropinocytosis through the uptake of FITC-dextran and amiloride inhibition of Francisella LVS uptake. Our results suggest that macropinocytosis is a potential mechanism of intracellular entry by LVS and that the host cell response is suppressed during the first 2-6 h of infection. These results suggest that the attenuated Francisella LVS induces significant host cell signaling at very early time points after the bacteria's interaction with the cell.

Antigen stored in dendritic cells after macropinocytosis is released unprocessed from late endosomes to target B cells.

B lymphocytes can be triggered in lymph nodes by nonopsonized antigens (Ag), potentially in their native form. However, the mechanisms that promote encounter of B lymphocytes with unprocessed antigens in lymph nodes are still elusive. We show here that antigens are detected in B cells in the draining lymph nodes of mice injected with live, but not fixed, dendritic cells (DCs) loaded with antigens. This highlights active processes in DCs to promote Ag transfer to B lymphocytes. In addition, antigen-loaded DCs found in the draining lymph node were CD103+. Using 3 different model Ag, we then show that immature DCs efficiently take up Ag by macropinocytosis and store the internalized material in late endocytic compartments. We find that DCs have a unique ability to release antigens from these compartments in the extracellular medium, which is controlled by Rab27. B cells take up the regurgitated Ag and the chemokine CXCL13, essential to attract B cells in lymph nodes, enhances this transfer. Our results reveal a unique property of DCs to regurgitate unprocessed Ag that could play an important role in B-cell activation.

Involvement of aquaporin-7 in the cutaneous primary immune response through modulation of antigen uptake and migration in dendritic cells.

Dendritic cells (DCs) have the ability to present antigen and play a critical role in the induction of the acquired immune response. Skin DCs uptake antigen and subsequently migrate to regional draining lymph nodes (LNs), where they activate naive T cells. Here we show that the water/glycerol channel protein aquaporin 7 (AQP7) is expressed on epidermal and dermal DCs and involved in the initiation of primary immune responses. AQP7-deficient DCs showed a decreased cellular uptake of low-molecular-mass compounds (fluorescein isothiocyanate and Lucifer yellow) and high-molecular-mass substances (ovalbumin and dextran), suggesting that AQP7 is involved in antigen uptake. AQP7-deficient DCs also exhibited reduced chemokine-dependent cell migration in comparison to wild-type DCs. Consistent with these in vitro results, AQP7-deficient mice demonstrated a reduced accumulation of antigen-retaining DCs in the LNs after antigen application to the skin, which could be attributed to decreased antigen uptake and migration. Coincidentally, AQP7-deficient mice had impaired antigen-induced sensitization in a contact hypersensitivity model. These observations suggested that AQP7 in skin DCs is primarily involved in antigen uptake and in the subsequent migration of DCs and is responsible for antigen presentation and the promotion of downstream immune responses.

Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis.

The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and induced ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies.

Uptake routes of tumor-antigen MAGE-A3 by dendritic cells determine priming of naïve T-cell subtypes.

Induction of tumor-antigen-specific T cells in active cancer immunotherapy is generally difficult due to the very low anti-tumoral precursor cytotoxic T cells. By improving tumor-antigen uptake and presentation by dendritic cells (DCs), this problem can be overcome. Focusing on MAGE-A3 protein, frequently expressed in many types of tumors, we analyzed different DC-uptake routes after additional coating the recombinant MAGE-A3 protein with either a specific monoclonal antibody or an immune complex formulation. Opsonization of the protein with antibody resulted in increased DC-uptake compared to the uncoated rhMAGE-A3 protein. This was partly due to Fcγ receptor-dependent internalization. However, unspecific antigen internalization via macropinocytosis also played a role. When analyzing DC-uptake of MAGE-A3 antigen expressed in multiple myeloma cell line U266, pretreatment with proteasome inhibitor bortezomib resulted in increased apoptosis compared to γ-irradiation. Bortezomib-mediated immunogenic apoptosis, characterized by elevated surface expression of hsp90, triggered higher phagocytosis of U266 cells by DCs involving specific DC-derived receptors. We further investigated the impact of antigen delivery on T-cell priming. Induction of CD8(+) T-cell response was favored by stimulating naïve T cells with either antibody-opsonized MAGE-A3 protein or with the bortezomib-pretreated U266 cells, indicating that receptor-mediated uptake favors cross-presentation of antigens. In contrast, CD4(+) T cells were preferentially induced after stimulation with the uncoated protein or protein in the immune complex, both antigen formulations were preferentially internalized by DCs via macropinocytosis. In summary, receptor-mediated DC-uptake mechanisms favored the induction of CD8(+) T cells, relevant for clinical anti-tumor response.

Hypoxia-induced macropinocytosis represents a metabolic route for liver cancer.

Hepatocellular carcinoma (HCC) invariably exhibits inadequate O2 (hypoxia) and nutrient supply. Hypoxia-inducible factor (HIF) mediates cascades of molecular events that enable cancer cells to adapt and propagate. Macropinocytosis is an endocytic process initiated by membrane ruffling, causing the engulfment of extracellular fluids (proteins), protein digestion and subsequent incorporation into the biomass. We show that macropinocytosis occurs universally in HCC under hypoxia. HIF-1 activates the transcription of a membrane ruffling protein, EH domain-containing protein 2 (EHD2), to initiate macropinocytosis. Knockout of HIF-1 or EHD2 represses hypoxia-induced macropinocytosis and prevents hypoxic HCC cells from scavenging protein that support cell growth. Germline or somatic deletion of Ehd2 suppresses macropinocytosis and HCC development in mice. Intriguingly, EHD2 is overexpressed in HCC. Consistently, HIF-1 or macropinocytosis inhibitor suppresses macropinocytosis and HCC development. Thus, we show that hypoxia induces macropinocytosis through the HIF/EHD2 pathway in HCC cells, harnessing extracellular protein as a nutrient to survive.

Cross-presentation of epitopes on virus-like particles via the MHC I receptor recycling pathway.

Effective vaccines and immunotherapies against cancer require professional antigen-presenting cells to cross-present exogenous antigen to initiate cytotoxic T-cell responses to destroy tumors. Virus-like particles (VLPs), containing tumor antigens, which can immunize against cancers, are cross-presented by dendritic cell (DC) but the mechanism by which this occurs is not fully understood. Here, we used VLPs, derived from rabbit hemorrhagic disease virus (RHDV) with both murine and human DCs, to elucidate these pathways. We have employed inhibitors to demonstrate that these VLPs are taken up by clathrin-dependent macropinocytosis and phagocytosis before being degraded in acidic lysosomal compartments. VLP-derived peptides are loaded onto major histocompatibility complex I that have been recycled from the cell surface. Antigen-coupled VLPs and murine ovalbumin-specific and human melanoma-associated antigen recognized by T cells (MART-1)-specific CD8(+) T cells were used to demonstrate cross-presentation via this alternate, receptor recycling pathway, which operated independently of the proteasome and the transporter-associated with antigen presentation. Finally, we found that cross-presentation of VLPs in vivo was not confined to CD8α(+) DC subsets. These data define the cross-presentation pathway for RHDV VLPs and may lead to improved cancer immunotherapies.

Activated neutrophil fluorescent imaging technique for human lungs.

Neutrophil activation is an integral process to acute inflammation and is associated with adverse clinical sequelae. Identification of neutrophil activation in real time in the lungs of patients may permit biological stratification of patients in otherwise heterogenous cohorts typically defined by clinical criteria. No methods for identifying neutrophil activation in real time in the lungs of patients currently exist. We developed a bespoke molecular imaging probe targeting three characteristic signatures of neutrophil activation: pinocytosis, phagosomal alkalinisation, and human neutrophil elastase (HNE) activity. The probe functioned as designed in vitro and ex vivo. We evaluated optical endomicroscopy imaging of neutrophil activity using the probe in real-time at the bedside of healthy volunteers, patients with bronchiectasis, and critically unwell mechanically ventilated patients. We detected a range of imaging responses in vivo reflecting heterogeneity of condition and severity. We corroborated optical signal was due to probe function and neutrophil activation.

Mesenchymal Stromal Cell Derived Membrane Particles Are Internalized by Macrophages and Endothelial Cells Through Receptor-Mediated Endocytosis and Phagocytosis.

Mesenchymal stromal cells (MSC) are a promising therapy for inflammatory diseases. However, MSC are large and become trapped in the lungs after intravenous infusion, where they have a short survival time. To steer MSC immunoregulatory therapy beyond the lungs, we generated nm-sized particles from MSC membranes (membrane particles, MP), which have immunomodulatory properties, and investigated their internalization and mode of interaction in macrophages subtypes and human umbilical vein endothelial cells (HUVEC) under control and inflammatory conditions. We found that macrophages and HUVEC take up MP in a dose, time, and temperature-dependent manner. Specific inhibitors for endocytotic pathways revealed that MP internalization depends on heparan sulfate proteoglycan-, dynamin-, and clathrin-mediated endocytosis but does not involve caveolin-mediated endocytosis. MP uptake also involved the actin cytoskeleton and phosphoinositide 3-kinase, which are implicated in macropinocytosis and phagocytosis. Anti-inflammatory M2 macrophages take up more MP than pro-inflammatory M1 macrophages. In contrast, inflammatory conditions did not affect the MP uptake by HUVEC. Moreover, MP induced both anti- and pro-inflammatory responses in macrophages and HUVEC by affecting gene expression and cell surface proteins. Our findings on the mechanisms of uptake of MP under different conditions help the development of target-cell specific MP therapy to modulate immune responses.

Cancer cells escape autophagy inhibition via NRF2-induced macropinocytosis.

Many cancers, including pancreatic ductal adenocarcinoma (PDAC), depend on autophagy-mediated scavenging and recycling of intracellular macromolecules, suggesting that autophagy blockade should cause tumor starvation and regression. However, until now autophagy-inhibiting monotherapies have not demonstrated potent anti-cancer activity. We now show that autophagy blockade prompts established PDAC to upregulate and utilize an alternative nutrient procurement pathway: macropinocytosis (MP) that allows tumor cells to extract nutrients from extracellular sources and use them for energy generation. The autophagy to MP switch, which may be evolutionarily conserved and not cancer cell restricted, depends on activation of transcription factor NRF2 by the autophagy adaptor p62/SQSTM1. NRF2 activation by oncogenic mutations, hypoxia, and oxidative stress also results in MP upregulation. Inhibition of MP in autophagy-compromised PDAC elicits dramatic metabolic decline and regression of transplanted and autochthonous tumors, suggesting the therapeutic promise of combining autophagy and MP inhibitors in the clinic.

Rab10-Positive Tubular Structures Represent a Novel Endocytic Pathway That Diverges From Canonical Macropinocytosis in RAW264 Macrophages.

Using the optogenetic photo-manipulation of photoactivatable (PA)-Rac1, remarkable cell surface ruffling and the formation of a macropinocytic cup (premacropinosome) could be induced in the region of RAW264 macrophages irradiated with blue light due to the activation of PA-Rac1. However, the completion of macropinosome formation did not occur until Rac1 was deactivated by the removal of the light stimulus. Following PA-Rac1 deactivation, some premacropinosomes closed into intracellular macropinosomes, whereas many others transformed into long Rab10-positive tubules without forming typical macropinosomes. These Rab10-positive tubules moved centripetally towards the perinuclear Golgi region along microtubules. Surprisingly, these Rab10-positive tubules did not contain any endosome/lysosome compartment markers, such as Rab5, Rab7, or LAMP1, suggesting that the Rab10-positive tubules were not part of the degradation pathway for lysosomes. These Rab10-positive tubules were distinct from recycling endosomal compartments, which are labeled with Rab4, Rab11, or SNX1. These findings suggested that these Rab10-positive tubules may be a part of non-degradative endocytic pathway that has never been known. The formation of Rab10-positive tubules from premacropinosomes was also observed in control and phorbol myristate acetate (PMA)-stimulated macrophages, although their frequencies were low. Interestingly, the formation of Rab10-positive premacropinosomes and tubules was not inhibited by phosphoinositide 3-kinase (PI3K) inhibitors, while the classical macropinosome formation requires PI3K activity. Thus, this study provides evidence to support the existence of Rab10-positive tubules as a novel endocytic pathway that diverges from canonical macropinocytosis.

C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells.

Removal of apoptotic cells is essential for maintenance of tissue homeostasis, organogenesis, remodeling, development, and maintenance of the immune system, protection against neoplasia, and resolution of inflammation. The mechanisms of this removal involve recognition of the apoptotic cell surface and initiation of phagocytic uptake into a variety of cell types. Here we provide evidence that C1q and mannose binding lectin (MBL), a member of the collectin family of proteins, bind to apoptotic cells and stimulate ingestion of these by ligation on the phagocyte surface of the multifunctional protein, calreticulin (also known as the cC1qR), which in turn is bound to the endocytic receptor protein CD91, also known as the alpha-2-macroglobulin receptor. Use of these proteins provides another example of apoptotic cell clearance mediated by pattern recognition molecules of the innate immune system. Ingestion of the apoptotic cells through calreticulin/CD91 stimulation is further shown to involve the process of macropinocytosis, implicated as a primitive and relatively nonselective uptake mechanism for C1q- and MBL-enhanced engulfment of whole, intact apoptotic cells, as well as cell debris and foreign organisms to which these molecules may bind.

Legionella pneumophila is internalized by a macropinocytotic uptake pathway controlled by the Dot/Icm system and the mouse Lgn1 locus.

The products of the Legionella pneumophila dot/icm genes enable the bacterium to replicate within a macrophage vacuole. This study demonstrates that the Dot/Icm machinery promotes macropinocytotic uptake of L. pneumophila into mouse macrophages. In mouse strains harboring a permissive Lgn1 allele, L. pneumophila promoted formation of vacuoles that were morphologically similar to macropinosomes and dependent on the presence of an intact Dot/Icm system. Macropinosome formation appeared to occur during, rather than after, the closure of the plasma membrane about the bacterium, since a fluid-phase marker preloaded into the macrophage endocytic path failed to label the bacterium-laden macropinosome. The resulting macropinosomes were rich in GM1 gangliosides and glycosylphosphatidylinositol-linked proteins. The Lgn1 allele restrictive for L. pneumophila intracellular replication prevented dot/icm-dependent macropinocytosis, with the result that phagosomes bearing the microorganism were targeted into the endocytic network. Analysis of macrophages from recombinant inbred mouse strains support the model that macropinocytotic uptake is controlled by the Lgn1 locus. These results indicate that the products of the dot/icm genes and Lgn1 are involved in controlling an internalization route initiated at the time of bacterial contact with the plasma membrane.

PolyI:C-induced reduction in uptake of soluble antigen is independent of dendritic cell activation.

Dendritic cells (DC) are key players in the initiation and modulation of adaptive immune responses due to their ability to acquire and present antigen and stimulate T cells. For the induction of effector T cell functions, antigen must be presented by activated DC. In this study, we have compared uptake of antigen by mouse DC in the presence of different Toll-like receptor (TLR) agonists, which are potent inducers of DC activation. Here we show that the reduction in uptake of soluble antigen in the presence of the viral double-stranded RNA (dsRNA) analogues polyinosinic-polycytidylic acid and Ampligen is independent of TLR-mediated DC activation. A reduction in antigen uptake by bone marrow-derived and splenic DC was also observed in response to other RNA homopolymers such as polyinosinic and polyguanylic acids, which are known inhibitors of scavenger receptor-mediated endocytosis. Pinocytosis and mannose receptor-mediated uptake of soluble antigen were not affected by any of the tested nucleic acids. The reduction in antigen uptake by dsRNA did not negatively influence the T cell stimulating properties of the DC. In summary, we conclude that the decrease in antigen endocytosis observed in the presence of a variety of TLR agonists is independent of TLR signalling and is caused by competition for specific surface receptors that are involved in the uptake of these TLR agonists and the antigen.

Aqueous extract of Phyllanthus niruri (Euphorbiaceae) enhances the phenotypic and functional maturation of bone marrow-derived dendritic cells and their antigen-presentation function.

Decoctions of Phyllanthus niruri (PN) (Fam. Euphorbiaceae) is promoted in traditional medicine of Africa, Asia, and South America as beneficial supplement for different infectious diseases, especially for viral hepatitis, tumor, and for immune compromised patients. This stimulated the interest in understanding the mechanisms by which the whole extract of the plant could stimulate the immune system. Dendritic cells (DCs) are professional antigen-presenting cells and provide a link between the innate and the adaptive immune responses. In the present study, the effects of lyophilized aqueous extract of PN on structural and functional maturation of murine bone marrow-derived DCs (BM-DCs) were investigated. Bone marrow cells were cultured in the presence of granulocyte macrophage-colony stimulating factor and interleukin-4 (IL-4) and the generated immature DCs were stimulated with PN (25, 50, and 100 microg/mL) or lipopolysaccharide (10 microg/mL) for 48 h. Results showed that treatment with PN increased the expression of major histocompatibility complex-II and the various makers for DCs maturation (CD40), activation (CD83), and costimulation (CD86) in a concentration-dependent manner. Consistent with the increase in phenotypic makers, functional maturation assay showed that treatment of BM-DCs with PN caused a decrease in fluorescein isothiocyanate-dextran pinocytosis and an increase in IL-12 in the supernatant. In a transgenic T-cell activation model, PN-treated BM-DCs presented Ova antigen to Ova-specific CD8(+) T cells from OT-1 mice more efficiently as demonstrated by increased T-cells proliferation and IL-2 production. Therefore, PN enhances the structural and functional maturation of BM-DCs and their antigen-presenting function. These effects are relevant in immunodeficient conditions, tumor control, and in infectious diseases.